Bi- and monospecific, asymmetric antibodies and methods of generating the same

ABSTRACT

An antibody is provided. The antibody comprises an Fc region and a Fab region, wherein:
         (i) the Fc region comprises two non-identical heavy chains, wherein at least one of the two non-identical heavy chains comprises an amino acid modification so as to form complementation between the two non-identical heavy chains thereby increasing the probability of forming heterodimers of the non-identical heavy chains and decreasing the probability of forming homodimers of identical heavy chains; and   (ii) the Fab region comprises a first covalent link between a first heavy chain and a first light chain of the Fab region and a second covalent link between a second heavy chain and a second light chain of said Fab region, wherein a position of the first covalent link relative to the first heavy chain is different to a position of the second covalent link relative to the second heavy chain.       

     Methods of generating same and uses thereof are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/005,580, filed on Sep. 17, 2013, which is a National Phase of PCTPatent Application No. PCT/IL2012/050093 having International FilingDate of Mar. 15, 2012, which claims the benefit of priority of U.S.Provisional Patent Application No. 61/453,591, filed on Mar. 17, 2011.The contents of the above applications are all incorporated by referenceas if fully set forth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 69273SequenceListing.txt, created on Feb. 22,2017, comprising 159,727 bytes, submitted concurrently with the filingof this application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates tobispecific antibodies, monospecific, asymmetric antibodies and methodsof generating same.

Bispecific antibodies (BsAbs) are antibodies with two binding sites,each directed against a different target antigen, to which they can bindsimultaneously (Baeuerle and Reinhardt, 2009). This property enables thedevelopment of therapeutic strategies that are not possible withconventional monoclonal antibodies. The primary applications ofbispecific antibodies include a) simultaneous inhibition of two targets(e.g. receptors of soluble ligands, a receptor and a ligand or twodifferent ligands), b) retargeting, where one binding specificity isdirected against a target cell (usually a tumor cell) whereas the otherbinding site is used to recruit a toxic activity or moiety to the targetcell (T or NK cells; enzyme for prodrug activation; cytokine,radionuclide, virus, toxin), c) increased specificity, when strongbinding mediated by simultaneous engagement of both antibody arms canonly occur on cells expressing both antigens (Fischer and Leger, 2007;Amann et al., 2009; Lutterbuese et al., 2010). Since bispecificantibodies are regarded as promising therapeutic agents, severalbispecific modalities have been developed, but their utility is limiteddue to problems with stability and manufacturing complexity. Severalstrategies for the creation of bispecific antibodies have been proposedover the past 20 years but despite numerous attempts and variousproposed antibody formats, the BsAbs suffer from lack of producthomogeneity and challenging production problems (Fischer and Leger,2007; Chames and Baty, 2009).

Initially, attempts were made to produce bispecific antibodies by fusingtwo hybridomas, each producing a different antibody, resulting in whatwas referred to as “quadromas” or hybrid hybridomas. However, quadromassuffered from genetic instability and yielded heterogeneous mixes of theheavy and light chains. It was found that on average an at randomassociation of L chains with H chains was found of the two antibodies,and only a tiny fraction were the desired bispecific antibodies (De Lauet al., 1991; Massino et al., 1997). If one considers creating abispecific antibody from two monospecific antibodies, A and B, efficientassembly of a bispecific antibody in an IgG format has two basicrequirements, one is that each heavy chain associates with the heavychain of the second antibody (heavy chain A associates with heavy chainB) and no homoassociation (A+A or B+B) occurs. The second requirement isthat each light chain associates with its cognate heavy chain (lightchain A with heavy chain A, and not light chain B with heavy chain A orlight chain A with heavy chain B). The random association of antibodychains in quadromas could not meet those requirements.

Efficient generation of bispecific antibodies was made possible byadvances in antibody engineering. Advanced antibody engineering enabledthe creation of new recombinant antibody formats like tandemsingle-chain variable fragment (scFv) (Robinson et al., 2008), diabodies(Hudson and Kortt, 1999), tandem diabodies (Kipriyanov, 2009),two-in-one antibody (Bostrom et al., 2009), and dual variable domainantibodies (Wu et al., 2007). These new antibody formats solved some ofthe manufacturing issues, providing homogeneous preparations. However,most of these scaffolds, due to their small size, suffer from poorpharmacokinetics and therefore require frequent dosing or conjugation tolarger carrier molecules to improve half-life (Constantinou et al.,2009).

Ridgway et al., 1996 provided a solution to one of the two criteria formaking bispecific antibodies making it possible to re-consider IgG-basedbispecific antibodies technically feasible. They described anengineering approach termed “knobs into holes” which allows onlyheterodimerization between the heavy chains of “antibodies A and B” toform, disallowing homodimerization. While studying the rules for heavychain association, the authors postulated that it is primarily dependenton interfacial interactions between the C_(H)3 domains of the two heavychains. When protein domains or subdomain interact, a knob is a bulkyside chains that protrudes into the opposite domain where it is alignedwith a small side chain that makes such invasion possible. In theirapproach, knob and hole variants were anticipated to heterodimerize byvirtue of the knob inserting into an appropriately designed hole on thepartner C_(H)3 domain. Knobs were constructed by replacing small sidechains with the largest side chains, tyrosine or tryptophan. Holes ofidentical or similar size to the knobs were created by replacing largeside chains with the smaller ones, in this case alanine or threonine.This way, two heavy chains that are knob variants can not homoassociatebecause of side chain clashes, and the homoassociation of two holevariants is less favored because of the absence of a stabilizingside-chain interaction. Subsequently, this group engineered a disulfidebond near the c-terminus of the CH3 domain to further stabilize theassembled bispecific antibodies (Merchant et al., 1998).

U.S. Pat. No. 7,183,076 teaches a method of generating bifunctionalantibodies using the knob and hole approach.

However, the knobs into holes approach provided a solution only for theheteroassociation of the heavy chain and did not provide one for thecorrect pairing of each heavy chain with its cognate light chain.Therefore, in that study, a bispecific IgG capable of simultaneouslybinding to the human receptors HER3 and cMpI was prepared bycoexpressing a common light chain and the corresponding remodeled heavychains followed by protein A chromatography. The engineered heavy chainsretain their ability to support antibody-dependent cell-mediatedcytotoxicity as demonstrated with an anti-HER2 antibody (Merchant etal., 1998).

International application 2010/115589 teaches trivalent bispecificantibodies in which to a monospecific IgG carrying knobs into holesmutations, a V_(H) and V_(L) of a second specificity are fused at theC-terminus of the two CH3 domains.

Similar molecules are described in U.S. Patent Application PublicationNo. 2010/0256340.

Disulfide-stabilized Fvs were first described by the group of AndreasPlückthun (Glockshuber et al., 1990) and later by the group of IraPastan (Brinkmann et al., 1993; Reiter et al., 1994a; Reiter et al.,1994b; Reiter et al., 1995). The Pastan group did extensive work ondsFvs, and used molecular modeling to identify positions in conservedframework regions of antibody Fv fragments (Fvs) that are distant fromCDRs, and potentially can be used to make recombinant Fv fragments inwhich the unstable V_(H) and V_(L) heterodimer is stabilized by anengineered interchain disulfide bond inserted between structurallyconserved framework positions. A disulfide bond was introduced at one ofthese positions, V_(H)44-V_(L)105 or V_(H)111-V_(L)48 was shown tostabilize various Fvs that retain full binding and specificity.

U.S. Pat. Nos. 5,747,654, 6,147,203 and 6,558,672 teachdisulfide-stabilized Fvs, wherein the Fvs are engineered to introduceadditional disulfide bonds between the light and heavy chains.

Additional background art includes Jackman et al., Journal of BiologicalChemistry Vol 285, No. 27, pp. 20850-20859, Jul. 2, 2010 and Schaefer etal., Proc Natl Acad Sci USA. 2011 July 5; 108(27): 11187-11192.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided an antibody comprising an Fc region and a Fab region,wherein:

(i) the Fc region comprises two non-identical heavy chains, wherein atleast one of the two non-identical heavy chains comprises an amino acidmodification so as to form complementation between the two non-identicalheavy chains thereby increasing the probability of forming heterodimersof the non-identical heavy chains and decreasing the probability offorming homodimers of identical heavy chains; and

(ii) the Fab region comprises a first covalent link between a firstheavy chain and a first light chain of the Fab region and a secondcovalent link between a second heavy chain and a second light chain ofthe Fab region, wherein a position of the first covalent link relativeto the first heavy chain is different to a position of the secondcovalent link relative to the second heavy chain.

According to an aspect of some embodiments of the present inventionthere is provided a method of preparing an antibody, comprising:

(a) providing a first nucleic acid molecule encoding the first heavychain;

(b) providing a second nucleic acid molecule encoding the second heavychain;

(c) providing a third nucleic acid molecule encoding the first lightchain;

(d) providing a fourth nucleic acid molecule encoding the second lightchain;

(e) culturing host cells comprising the first, second, third and fourthnucleic acid molecules under conditions that permit expression of thenucleic acid molecules; and

(f) recovering the antibody.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as an activeagent the antibody disclosed herein and a pharmaceutically acceptablecarrier.

According to an aspect of some embodiments of the present inventionthere is provided an antibody for treating an infection or inflammatorydisease or disorder.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating an infection or an inflammatorydisease or disorder in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of theantibody disclosed herein, thereby treating the infection orinflammatory disease or disorder.

According to some embodiments of the invention, the antibody is abispecific antibody.

According to some embodiments of the invention, the antibody is anasymmetric, mono specific antibody.

According to some embodiments of the invention, the complementationcomprises a steric complementation.

According to some embodiments of the invention, the complementationcomprises a charge complementation.

According to some embodiments of the invention, the Fc region comprisesa protuberance of one heavy chain of the Fc region and a stericallycompensatory cavity on a second heavy chain of the Fc region, theprotuberance protruding into the compensatory cavity.

According to some embodiments of the invention, the protuberance isgenerated by substituting an amino acid at one position on a CH3 domainof the one heavy chain with another amino acid having a larger sidechain volume than the original amino acid.

According to some embodiments of the invention, the compensatory cavityis generated by substituting an amino acid at one position on a CH3domain of the second heavy chain with another amino acid having asmaller side chain volume than the original amino acid.

According to some embodiments of the invention, the first covalent linkis between a CH1 domain of the one heavy chain and a CL domain of theone light chain; and the second covalent link is between a V_(H) domainof the second heavy chain and a V_(L) domain of the second light chain.

According to some embodiments of the invention, the first and the secondcovalent links are disulfide bonds.

According to some embodiments of the invention, the amino acid having alarger side chain volume than the original amino acid is selected fromthe group consisting of tyrosine, arginine, phenylalanine, isoleucineand tryptophan.

According to some embodiments of the invention, the amino acid having asmaller side chain volume than the original amino acid is selected fromthe group consisting of alanine, glycine, valine and threonine.

According to some embodiments of the invention, the antibody is selectedfrom the group consisting of a chimeric antibody, a humanized antibodyand a fully human antibody.

According to some embodiments of the invention, the CH3 domain of thefirst heavy chain is covalently linked to the CH3 domain of the secondheavy chain.

According to some embodiments of the invention, the first antigenbinding site of the antibody binds a first epitope of an antigen and thesecond antigen binding site of the antibody binds a second epitope ofthe antigen.

According to some embodiments of the invention, the first antigenbinding site of the antibody binds an epitope of a first antigen and thesecond antigen binding site of the antibody binds an epitope of a secondantigen.

According to some embodiments of the invention, each light chain islinked to its cognate heavy chain via a single disulfide bond.

According to some embodiments of the invention, the antibody is anintact antibody.

According to some embodiments of the invention, the antibody is selectedfrom the group consisting of IgA, IgD, IgE and IgG.

According to some embodiments of the invention, the IgG comprises IgG1,IgG2, IgG3 or IgG4.

According to some embodiments of the invention, the first heavy chaincomprises a T366W mutation; and the second heavy chain comprises T366S,L368A, Y407V mutations.

According to some embodiments of the invention, the first heavy chaincomprises an S354C mutation and the second heavy chain comprises a Y349Cmutation.

According to some embodiments of the invention, the first antigenbinding site binds CD30 and the second antigen binding site binds erbB2.

According to some embodiments of the invention, the first antigenbinding site binds CD30 and the second antigen binding site bindsPseudomonas Exotoxin (PE).

According to some embodiments of the invention, the first antigenbinding site binds CD30 and the second antigen binding site bindsstrepavidin.

According to some embodiments of the invention, at least one of theheavy chains is attached to a therapeutic moiety.

According to some embodiments of the invention, at least one of theheavy chains is attached to an identifiable moiety.

According to some embodiments of the invention, the antibody is selectedfrom the group consisting of a primate antibody, a porcine antibody, amurine antibody, a bovine antibody, a goat antibody and an equineantibody.

According to some embodiments of the invention, the host cells comprisebacterial cells.

According to some embodiments of the invention, the host cells comprisemammalian cells.

According to some embodiments of the invention, the expression takesplace in inclusion bodies of the bacterial cells.

According to some embodiments of the invention, each of the nucleic acidmolecules are transfected into different host cells.

According to some embodiments of the invention, each of the nucleic acidmolecules are transfected into the same host cell.

According to some embodiments of the invention, the bacterial cellscomprise gram negative bacterial cells.

According to some embodiments of the invention, the method furthercomprises purifying the antibody on Protein A/G/L following step (f).

According to some embodiments of the invention, the inflammatorydisorder is cancer.

According to some embodiments of the invention, the inflammatory diseaseor disorder is cancer.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-1B are schematic structure of a novel strategy for productionof bispecific antibodies. (A) scheme of an IgG antibody produced by theknobs into holes approach, there are two different heavy chains but acommon light chain. (B) scheme of a bispecific antibody preparedaccording to embodiments of the present invention. There are twodifferent heavy chains, each paired to its cognate light chain. The“knob” mutation corresponds to T366W, the “hole” mutations correspond toT366S, L368A Y407V replacements. Cysteine replacement mutations S354Cand Y349C at CH3 region of “knob” or “hole” heavy chain, respectively,provide 95% heterodimerization (Merchant et al., 1998).

FIGS. 2A-2H are schematic representation of pHAK-IgH- and pHAK-IgL-basedplasmid maps for expression of mono- and bispecific antibodies in E.coli: pHAK-IgL for expression of antibodies with human κ or λ lightchain, pHAK-LC-Cys for expression of light chains containing dsFv-likeintrachain disulfide bond, pHAK-IgH for expression of antibodies withhuman γ1 heavy chain, pHAK-HC-knob for expression of heavy chaincontaining S354C and T366W “knob” mutations in the constant region,pHAK-HC-hole for expression of heavy chain containing Y349C, T366S,L368A and Y407V “hole” mutation in constant region, pHAK-HC-hole-PE38for expression of heavy chain containing “hole” mutations fused to atruncated form of Pseudomonas exotoxin A (PE38), pHAK-HC-Cys forexpression of heavy chain containing dsFv-like disulfide intrachainbond, pHAK-HC-Cys-knob for expression of heavy chain containing “knob”mutations in constant region and dsFv-like intrachain disulfide bond.

FIG. 3 is a photograph of SDS-PAGE analysis of heavy and light chainspurification. The expressed proteins were collected as inclusion bodies,purified by sequential centrifugation steps and dissolved in a 6Mguanidinium hydrochloride buffered solution. (1) T427 IgL, (2) T427 IgH,(3) T427-IgH-knob, (4) T427-IgH-PE38, (5) T427-IgH-hole-PE38. “Knob”mutations correspond to S354C:T366W. “Hole” mutations correspond toY349C:T366S:L368A:Y407V.

FIGS. 4A-4B provide analysis of bispecific IgG-like proteins. (A) Theschematic structures of IgG heavy and light chains and the theoreticallypossible IgG molecules that can be formed. Each variant can be easilydetected according the significant differences in molecular weight. (B)SDS-PAGE (10% acrylamide) analysis of protein A purified products: wtT427 antibody displaying PE38 on heavy chain (1), “knobs-into-holes”version (2) of T427 antibody (S354C:T366W/Y349C:T366S:L368A:Y407Vmutations), wt FRP5 antibody (3).

FIGS. 5A-5B provide SDS-PAGE (10% acrylamide) analysis of protein Apurified products. (1) T427 “knob-knob” version (IgL+IgH-knobS354C:T366W mutations). (2) “Knobs-into-holes” version of T427 antibody(S354C:T366W/Y349C:T366S:L368A:Y407V mutations). (3) T427 “hole-hole”version (IgL+IgH-hole-PE38 Y349C:T366S:L368A:Y407V mutations). (4) wtT427 antibody displaying PE38 on heavy chain. (M) Marker.

FIG. 6 shows a gel filtration analysis of IgG and IgG-like proteins.T427 IgG antibody (150 kDa) elutes the Sephadex 200 gel filtrationcolumn at 11.5 min. The IgG-like T427 heterodimer (2IgL+IgH-knob+IgH-hole-PE38), 190 kDa elutes at 10.3 min. The smallfraction of knob-knob homodimer (150 kDa) elutes at 11.5 min. Thehole-hole homodimer (230 kDa) probably elutes at void volume (6.5 min(not shown)).

FIGS. 7A-7C illustrate SDS-PAGE (7.5% acrylamide) and density analysisof protein A purified products. (A) SDS-PAGE analysis of T427 IgG wt(1), T427-knob-hole-PE38 (2) and T427-PE38 (3) proteins. (B) Proteinband density analysis of SDS-PAGE by ImageMaster 1D scanning laserdensitometry. (C) The pie chart of the heterodimerization yield wasmeasured according the pixel intensity at band position.T427-knob-hole-PE38 (2) consists of 2IgL+IgH-knob+IgH-hole-PE38.T427-PE38 (3) consists of 2IgL+2IgH-PE38. “Knob” mutations correspond toS354C:T366W. “Hole” mutations correspond to Y349C:T366S:L368A:Y407V.

FIGS. 8A-8B illustrate ELISA analysis of IgG and IgG-like proteins. Thebinding ability of FRP5 IgG and bispecific FRP5-T437-PE38 (PE38 fused toT427 heavy chain). (A) The ELISA plate was coated with erbB2 (antigen ofFRP5 antibody) and antibodies were detected with anti-human secondaryantibody. (B) The ELISA plate was coated with erbB2 (antigen of FRP5antibody) and antibodies were detected with anti-PE secondary antibody(detection of bispecific antibodies).

The FRP5-T427-PE38 antibody consists ofIgL-FRP5+IgL-T427+IgH-FRP5-knob+IgH-T427-hole-PE38 proteins.“Knobs-into-holes” mutations:

S354C:T366W/Y349C:T366S:L368A:Y407V.

FIGS. 9A-9B illustrate SDS-PAGE (12% and 6% acrylamide) analyses ofprotein A purified IgG and IgG-like proteins. (1) FRP5 IgG wt. (2) T427IgG wt. (3) T427 IgG-Cys (IgH-Cys44:Cys222Ala+IgL-Cys104:Cys218del). (4)Bispecific T427-FRP5 IgG(IgH-FRP5-hole+IgL-FRP5+IgH-T427-khob-Cys44:Cys222Ala+IgL-T427-Cys104:Cys218delIgG. “Knob” mutations correspond to S354C:T366W. “Hole” mutationscorrespond to Y349C:T366S:L368A:Y407V.

FIG. 10 is an SDS-PAGE (10% acrylamide) analysis of protein A purifiedIgG and IgG-like proteins. (1) T427 IgG wt. (2) Anti-Tac IgG wt. (3)T427 IgG-Cys control A (IgH wt+IgL-Cys104:Cys218del). (4) T427 IgG-Cyscontrol B (IgH-Cys44:Cys222Ala+IgL wt).

FIG. 11 is an SDS-PAGE analysis of heavy and light chains of αPE (B11),T427 and αSA antibodies purified as inclusion bodies and resuspended in6 M guanidinium hydrochloride. The samples were separated in reducingcondition on 12% acrylamide gel.

FIGs. 12A-12B is ELISA analysis of αSA (anti-streptavidin) antibodies.The T427, αSA (monoclonal) and T427-αSA (bispecific) protein A purifiedantibodies were analyzed for their ability to bind CD30 (A). The bindingwas detected using goat-anti-human HRP conjugated antibodies. Coatingwith bovine serum albumin (BSA) served as a control (B).

FIGS. 13A-13C is an ELISA analysis of αPE (anti-Pseudomonas exotoxin 38kDa fragment) antibodies. The T427, αPE B11 clone (monoclonal) andT427-αPE (bispecific) protein A purified antibodies were analyzed fortheir ability to bind avitag-PE38 (A) and dsFv-PE38 (B) antigens. Thebinding was detected using goat-anti-human HRP conjugated antibodies.Coating with bovine serum albumin (BSA) served as a control (C).

FIG. 14 is a schematic presentation of pDual vector system. pDualvectors are bi-cistronic, CMV promoter-based plasmids for the expressionof IgGs in mammalian cells. They were constructed by combining heavy andlight chain expression cassettes from the pMAZ vectors (Mazor Y, JImmunol Methods. 2007 Apr. 10; 321(1-2):41-59).

FIGS. 15A-15B illustrate analyses of secreted IgG in medium of CaCl₂transfected 293 Trex cells. (A) Western blot analysis of cell mediatransfected with pDual wt, pDual L(Cys)+H(wt) or pMAZ-IgL+pMAZ-IgHvectors systems. The antibodies were detected with goat-anti-human HRPconjugated secondary antibody.

The antibody concentration in media was determined in comparison to thesecondary dilutions of Erbitux antibody (B).

FIG. 16 illustrates a Western blot analysis of 293 Trex cellstransfected with pDual mono-specific and bispecific vectors orcombination of vectors. The antibodies were detected withgoat-anti-human HRP conjugated antibody.

FIG. 17 illustrates exemplary results from a Dot blot analysis ofantibody secreting clones. The cell media were absorbed tonitrocellulose membrane and antibodies were detected withgoat-anti-human HRP conjugated antibody. The secretion level wasdetermined relatively to other clones. The cell media of non-treatedcells served as control.

FIGS. 18A-B illustrate validation of binding activity of bispecificclones. The cell media were incubated with either erbB2 (18A) or CD30(18B) antigens. The binding was detected with goat-anti-human HRPconjugated antibody. The marked clones demonstrated the binding abilityto both antigens.

FIGS. 19A-19B illustrates an SDS-PAGE analysis of IgGs produced in HEK293 T-REx™ mammalian cells followed by protein A purification. Theproteins were separated in unreduced conditions on 10% acrylamide gel inorder to evaluate the 150 kDa IgGs (A) and in reduced conditions on 12%acrylamide gel (B) in order to evaluate the minimal differences betweenT427 and FRP5 heavy chains and light chains and determine the doublebands in bispecific T427-FRP5 molecules.

FIGS. 20A-20B illustrate ELISA analysis of protein A purified IgGsproduced in mammalian cells. A5 is a control cell line transfected withfour plasmids, two encoding the monospecific T427 antibody and twoencoding the monospecific FRP5 antibody. Bispecific T427-FRP5 representsbispecific antibody secreting stable clone D3 with mono-valent bindingability to each antigen (erbB2 and CD30). The binding was detected usinggoat-anti-human HRP conjugated secondary antibody.

FIGS. 21A-21B are graphs illustrating ELISA analysis of protein Apurified IgGs produced in mammalian cells. T427 and FRP5 representmono-specific antibodies with bi-valent binding activity. BispecificT427-FRP5 represents bispecific antibody-secreting stable clone D3 withmono-valent binding ability to each antigen (erbB2 and CD30). Erbituxserved as a negative control. The binding was detected usinggoat-anti-human HRP conjugated secondary antibody.

FIG. 22 is a graph illustrating cell-ELISA analysis of binding abilityof B3 clone secreting T427-FRP5 bispecific antibody, (protein A-purifiedfrom conditioned medium of the stable clone) to A431/CD30 and SKBR3(erbB2+) cells. The binding was detected using goat-anti-human HRPconjugated secondary antibody.

FIG. 23 is a schematic representation of the monospecific antibody ofembodiments of the present invention.

FIG. 24 is ELISA analysis of T427 KIH. The binding ability of T427 IgGand T427-PE38 (PE38 fused to heavy chain) in comparison toknobs-into-holes (KIH) version of T427 antibody(2×IgL+IgH-knob+IgH-hole-PE38).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates tobispecific antibodies, monospecific antibodies, asymmetric antibodiesand methods of generating same.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

In past years, both laboratory and early clinical studies havedemonstrated that bispecific antibodies (BsAbs) may have significantpotential application in cancer therapy either by targeting tumor cellswith cytotoxic agents including effector cells, radionuclides, drugs,and toxins, or by simultaneously blocking two relevant tumor targets,that is, growth factor receptors or their ligands, thus neutralizingmultiple receptor activation and downstream signal transductionpathways. A major obstacle in the development of BsAb has been thedifficulty of producing the materials in sufficient quality and quantityby traditional technologies such as the hybrid hybridoma and chemicalconjugation methods. Thus, it is believed that the development ofIgG-like BsAbs as therapeutic agents will depend heavily on the advancesmade in the design of recombinant BsAb constructs and productionefficiency.

In order to ensure heterodimerization between the heavy chains ofantibodies “A” and “B” to form, and to prevent homodimerization ofantibody “A” to antibody “A” and antibody “B” to antibody “B”, a knoband hole approach has been suggested, as disclosed in U.S. Pat. No.7,183,076. However, the knobs into holes approach provides a solutiononly for the heteroassociation of the heavy chains and does not provideone for the correct pairing of each heavy chain with its cognate lightchain.

The present invention relates to an approach of efficient assembly ofbispecific antibodies in an IgG format. The approach involvesheterodimerization of the two heavy chains by applying the knobs intoholes approach, combined with facilitation of pairing of each heavychain with only its cognate light chain.

The present inventors suggest pairing the heavy and light chains of thesame antibody using one native CH1-CL binding disulfide bond and onenon-native V_(H)—V_(L) binding dsFv-like di-sulfide bond (as illustratedin FIG. 1B). This way one antibody branch would stay molecularlyuntouched while the other antibody branch would acquire a new disulfidecovalent bond in variable region instead of the wt S—S bond. Themis-paired light and heavy chains would not form the S—S stabilizedinterface and would not produce stable IgG molecule. Thus this strategysupposes the conversion of one antibody branch into dsFv-like moleculewithout any affinity or stability loss.

Whilst reducing the present invention to practice, the present inventorsgenerated a bispecific antibody by combining an anti-CD30 (T427) and ananti-erbB2 (FRP5) antibody. In the erbB2 antibody, heavy-light chainassociation was facilitated by the natural disulfide bond thatcovalently links the C_(H)1 domain of the heavy chain with the C_(L)domain of the light chain. In the anti-CD30 antibody, the cysteine inC_(H)1 was mutated to alanine and the C-terminal cysteine of C_(L) wasdeleted, preventing the formation of the native H-L disulfide bond.Instead of the eliminated disulfide bond, two cysteines, one in thevariable domain of the heavy chain and one in the variable domain of thelight chain were introduced according the rules of disulfide-stabilizedFv fragments (dsFvs). As a result, the heavy and light chains of theanti-CD30 antibody associated covalently via a disulfide bond that formsbetween V_(H) and V_(L) through these two cysteine residues. Thus, thepresent invention contemplates both the generation of a novel disulfidebridge between the heavy chain and its cognate light chain on one arm ofthe bifunctional antibody and so as to further enhance correct assembly,deletion of the naturally occurring disulfide bridge between the sameheavy chain with its cognate light chain.

As illustrated in FIGS. 9A-B, using this approach, full-lengthbifunctional antibodies were generated in bacterial cells. When theheavy and light chains of the anti-CD30 antibody were not mutated asdescribed above, full length bifunctional antibodies were not generated(FIG. 10).

Further, using bispecific vectors, the present inventors showed that thegeneration of full-length bifunctional antibodies in mammalian cells waspromoted by applying the knobs into holes approach, combined withfacilitation of pairing of each heavy chain with only its cognate lightchain (as illustrated in FIGS. 17-22).

Thus, according to an aspect of the present invention there is providedan antibody comprising an Fc region and a Fab region, wherein:

(i) the Fc region comprises two non-identical heavy chains, wherein atleast one of the two non-identical heavy chains comprises an amino acidmodification so as to form complementation between the two non-identicalheavy chains thereby increasing the probability of forming heterodimersof the non-identical heavy chains and decreasing the probability offorming homodimers of identical heavy chains; and

(ii) the Fab region comprises a first covalent link between a firstheavy chain and a first light chain of the Fab region and a secondcovalent link between a second heavy chain and a second light chain ofthe Fab region, wherein a position of the first covalent link relativeto the first heavy chain is different to a position of the secondcovalent link relative to the second heavy chain.

An antibody is characterized by a centrally placed disulfide bridge thatstabilizes a series of antiparallel beta strands into animmunoglobulin-like fold. An antibody heavy or light chain has anN-terminal (NH₂) variable region (V), and a C-terminal (—COOH) constantregion (C). The heavy chain variable region is referred to as V_(H), andthe light chain variable region is referred to as V_(L). V_(H) and V_(L)fragments together are referred to as “Fv”. The variable region is thepart of the molecule that binds to the antibody's cognate antigen, whilethe constant region determines the antibody's effector function (e.g.,complement fixation, opsonization). Full-length immunoglobulin orantibody “light chains” (generally about 25 kilodaltons (Kd), about 214amino acids) are encoded by a variable region gene at the N-terminus(generally about 110 amino acids) and a constant region gene at theCOOH-terminus. Full-length immunoglobulin or antibody “heavy chains”(generally about 50 Kd, about 446 amino acids), are similarly encoded bya variable region gene (generally encoding about 116 amino acids) andone of the constant region genes (encoding about 330 amino acids). Anantibody light or heavy chain variable region comprises threehypervariable regions, also called complementarity determining regionsor CDRs, flanked by four relatively conserved framework regions or FRs.

According to one embodiment of this aspect of the present invention theantibody is a bispecific antibody.

As used herein, the term “bispecific antibody” refers to an antibodywhich comprises two antigen binding sites, each binding to a differentepitope of an antigen. The bispecific antibodies of this aspect of thepresent invention do not share common light chains nor common heavychains.

According to one embodiment, the two antigen binding sites each bind todifferent epitopes of an identical antigen. According to anotherembodiment, the two antigen binding sites each bind to differentepitopes on different antigens.

According to another embodiment of this aspect of the present invention,the antibody is a monospecific, asymmetric antibody.

The monospecific antibodies of this aspect of the present invention havethe same paratope on both arms which bind an identical antigen. However,unlike conventional monoclonal antibodies which are symmetric assembliesof two identical heavy chains and two identical light chains,monospecific antibodies described herein are asymmetric assemblies oftwo non-identical heavy chains and two non-identical light chains. Thedifferences between the two heavy chains and between the two lightchains are in the constant domains and in framework regions of thevariable domains that allow heterodimerization of the chains.Accordingly, the CDR loops of the variable domains and supportingvariable domain residues that may comprise the paratope are identical inthe chain pairs—see FIG. 23.

According to a particular embodiment, the monospecific antibody is anIgG4.

Preferably, the affinity of each of the antigen binding sites of theantibody for its target is not substantially reduced as compared withone arm of its corresponding monoclonal antibody for the identicaltarget. According to a specific embodiment, the affinity is not reducedmore than 100 fold, more preferably is not reduced more than 50 fold,more preferably is not reduced more than 20 fold, more preferably is notreduced more than 10 fold and even more preferably is not reduced morethan 5 fold.

Examples of bispecific antibodies include those with one antigen bindingsite directed against a first growth factor ligand and a second antigenbinding site directed against a second growth factor ligand; one antigenbinding site directed against a first growth factor receptor and asecond antigen binding site directed against a second growth factorreceptor; one antigen binding site directed against a first cytokine anda second antigen binding site directed against a second cytokine; oneantigen binding site directed against a first cytokine receptor and asecond antigen binding site directed against a second cytokine receptor;one antigen binding site directed against a growth factor receptor and asecond antigen binding site directed against a growth factor ligand; oneantigen binding site directed against a cytokine receptor and a secondantigen binding site directed against a cytokine ligand. Additionalcombinations of growth factors, growth factor receptors, cytokines andcytokine receptors are also contemplated.

According to another embodiment, the bispecific antibody block twopathways of angiogenesis, one antigen binding site is directed towards areceptor or ligand associated with the first pathway and the otherantigen binding site is directed towards a receptor or ligand associatedwith the second pathway.

According to a specific embodiment, the bispecific antibody comprisesone antigen binding site directed against a tumor cell antigen and theother antigen binding site directed against a cytotoxic trigger moleculesuch as anti-Fcγ-RI/anti-CD15, anti-p185^(HER2)/Fcγ-RIII (CD16),anti-CD3/anti-malignant B-cell (1D10), anti-CD 3/anti-p185^(HER2),anti-CD3/anti-p97, anti-CD3/anti-renal cell carcinoma,anti-CD3/anti-OVCAR-3, anti-CD3/L-D1 (anti-colon carcinoma),anti-CD3/anti-melanocyte stimulating hormone analog, anti-EGFreceptor/anti-CD3, anti-CD3/anti-CAMA1, anti-CD3/anti-CD19,anti-CD3/MoV18, anti-neural cell adhesion molecule (NCAM)/anti-CD3,anti-folate binding protein (FBP)/anti-CD3, anti-pan carcinomaassociated antigen (AMOC-31)/anti-CD3.

Bispecific antibodies with one antigen binding site binding specificallyto a tumor antigen and one antigen binding site binding to a toxininclude for example anti-saporin/anti-Id-1, anti-CD22/anti-saporin,anti-CD7/anti-saporin, anti-CD38/anti-saporin, anti-CEA/anti-ricin Achain, anti-interferon-α (IFN-α)/anti-hybridoma idiotype,anti-CEA/anti-vinca alkaloid.

Other contemplated bispecific antibodies include those for convertingenzyme activated prodrugs such as anti-CD30/anti-alkaline phosphatase(which catalyzes conversion of mitomycin phosphate prodrug to mitomycinalcohol).

Other contemplated bispecific antibodies include those which can be usedas fibrinolytic agents such as anti-fibrin/anti-tissue plasminogenactivator (tPA), anti-fibrin/anti-urokinase-type plasminogen activator(uPA).

Additional contemplated bispecific antibodies include those fortargeting immune complexes to cell surface receptors such as anti-lowdensity lipoprotein (LDL)/anti-Fc receptor (e.g. Fcγ-RI, Fcγ-RII orFcγ-RIII).

Additional contemplated bispecific antibodies include those for use intherapy of infectious diseases such as anti-CD3/anti-herpes simplexvirus (HSV), anti-T-cell receptor:CD3 complex/anti-influenza,anti-FcγR/anti-HIV. Further bispecific antibodies for tumor detection invitro or in vivo include anti-CEA/anti-EOTUBE, anti-CEA/anti-DPTA,anti-p185HER2/anti-hapten.

Bispecific antibodies may be used as vaccine adjuvants (see Fanger etal., Critical Reviews in Immunology 12(3,4):101-124 (1992)).

Bispecific antibodies may be used as diagnostic tools such asanti-rabbit IgG/anti-ferritin, anti-horse radish peroxidase(HRP)/anti-hormone, anti-somatostatin/anti-substance P,anti-HRP/anti-FITC, anti-CEA/anti-β-galactosidase.

Additional contemplated bispecific antibodies include ones where thefirst antigen binding site binds CD30 and the second antigen bindingsite binds erbB2; ones where the first antigen binding site binds CD30and the second antigen binding site binds Pseudomonas Exotoxin (PE);ones where the first antigen binding site binds CD30 and the secondantigen binding site binds Streptavidin.

Examples of trispecific antibodies include anti-CD3/anti-CD4/anti-CD37,anti-CD3/anti-CD5/anti-CD37 and anti-CD3/anti-CD8/anti-CD37.

The Fc region of the antibodies of the present invention may be may beobtained from any antibody, such as IgG₁, IgG₂, IgG₃, or IgG₄ subtypes,IgA, IgE, IgD or IgM.

According to one embodiment, the Fc region is an IgG Fc region.

As mentioned, the Fc region of the antibodies described herein comprisestwo non-identical heavy chains (e.g. that differ in the sequence of thevariable domains), wherein at least one of the two non-identical heavychains comprises an amino acid modification so as to increase theprobability of forming a stable heterodimer of the non-identical heavychains and decrease the probability of forming a stable homodimer ofidentical heavy chains.

According to one embodiment, at least one heavy chain is geneticallymodified such that an altered charge polarity across the interface iscreated. As a consequence, a stable heterodimer betweenelectrostatically matched Fc chains is promoted, and unwanted Fchomodimer formation is suppressed due to unfavorable repulsive chargeinteractions.

Determination of which amino acids to modify and to which amino acids isfurther explained in Gunasekaran K, Pentony M, Shen M, Garrett L, ForteC, Woodward A, Ng S B, Born T, Retter M, Manchulenko K, Sweet H, Foltz IN, Wittekind M, Yan W. Enhancing antibody Fc heterodimer formationthrough electrostatic steering effects: applications to bispecificmolecules and monovalent IgG. J Biol Chem. 2010 Jun. 18;285(25):19637-46. Epub 2010 Apr. 16, incorporated herein by reference.

According to one embodiment, the amino acid modifications (that affectcharge complementarity) are effected at the rim of the interface betweenthe two heavy chains and not in structurally conserved buried residuesat the hydrophobic core of the interface.

According to another embodiment, at least one heavy chain is geneticallymodified, to generate a heavy chain with a 3D structure which binds moreefficiently to the non-identical heavy chain (i.e. a heterodimer) asopposed to an identical heavy chain (i.e. a homodimer). The generationof heterodimers is encouraged due to steric complementation and thegeneration of homodimers is discouraged due to steric hindrance.

According to this embodiment, one heavy chain is genetically modified togenerate a protuberance and the second heavy chain is geneticallymodified to generate a sterically compensatory cavity, the protuberanceprotruding into the compensatory cavity.

“Proturbances” are constructed by replacing small amino acid side chainsfrom the interface of the first heavy chains with larger side chains(e.g. tyrosine, arginine, phenylalanine, isoleucine, leucine ortryptophan). Compensatory “cavities” of identical or similar size to theprotuberances are optionally created on the interface of the secondheavy chain by replacing large amino acid side chains with smaller ones(e.g. alanine, glycine, serine, valine, or threonine).

The protuberance or cavity can be “introduced” into the interface of thefirst or second heavy chain by synthetic means, e.g. by recombinanttechniques, in vitro peptide synthesis, those techniques for introducingnon-naturally occurring amino acid residues previously described, byenzymatic or chemical coupling of peptides or some combination of thesetechniques. According, the protuberance, or cavity which is “introduced”is “non-naturally occurring” or “non-native”, which means that it doesnot exist in nature or in the original polypeptide (e.g. a humanizedmonoclonal antibody).

Preferably the import amino acid residue for forming the protuberancehas a relatively small number of “rotamers” (e.g. about 3 6). A“rotamer” is an energetically favorable conformation of an amino acidside chain. The number of rotamers of the various amino acid residuesare reviewed in Ponders and Richards, J. Mol. Biol. 193:775 791 (1987).

As a first step to selecting original residues for forming theprotuberance and/or cavity, the three-dimensional structure of theantibodies are obtained using techniques which are well known in the artsuch as X-ray crystallography or NMR. Based on the three-dimensionalstructure, those skilled in the art will be able to identify theinterface residues.

The preferred interface is the C_(H3) domain of an immunoglobulinconstant domain. It is preferable to select “buried” residues to bereplaced. The interface residues of the CH3 domains of IgG, IgA, IgD,IgE and IgM have been identified (see, for example, PCT/US96/01598,herein incorporated by reference in its entirety), including those whichare optimal for replacing with import residues; as were the interfaceresidues of various IgG subtypes and “buried” residues. The preferredC_(H3) domain is derived from an IgG antibody, such as an human IgG₁.

The C_(H3)/C_(H3) interface of human IgG₁ involves sixteen residues oneach domain located on four anti-parallel β-strands which buries 1090ANG² from each surface. Mutations are preferably targeted to residueslocated on the two central anti-parallel β-strands. The aim is tominimize the risk that the protuberances which are created can beaccommodated by protruding into surrounding solvent rather than bycompensatory cavities in the partner C_(H3) domain. Methods of selectionparticular sites on the heavy chains have been disclosed in U.S. Pat.No. 7,183,076, incorporated herein by reference.

According to a specific embodiment, the first heavy chain comprises aT366W mutation (i.e. threonine to tryptophan); and the second heavychain comprises T366S, L368A, Y407V mutations (i.e. threonine to serine;leucine to alanine; and tyrosine to valine).

According to one embodiment, the amino acid modifications (that affectstructural complementarity) are effected at structurally conservedburied residues at the hydrophobic core of the interface, and not in atthe rim of the interface between the two heavy chains.

The effect of replacing residues on the heavy chains can be studiedusing a molecular graphics modeling program such as the Insight™ program(Biosym Technologies).

Once the preferred original/import residues are identified by molecularmodeling, the amino acid replacements may be introduced into the heavychains using techniques which are well known in the art.

Oligonucleotide-mediated mutagenesis is a preferred method for preparingsubstitution variants of the DNA encoding the first or second heavychain. This technique is well known in the art as described by Adelmanet al., DNA, 2:183 (1983). Briefly, first or second polypeptide-codingDNA is altered by hybridizing an oligonucleotide encoding the desiredmutation to a DNA template, where the template is the single-strandedform of a plasmid or bacteriophage containing the unaltered or nativeDNA sequence of heteromultimer. After hybridization, a DNA polymerase isused to synthesize an entire second complementary strand of the templatethat will thus incorporate the oligonucleotide primer, and will code forthe selected alteration in the heteromultimer DNA.

Cassette mutagenesis can be performed as described Wells et al. Gene34:315 (1985) by replacing a region of the DNA of interest with asynthetic mutant fragment generated by annealing complimentaryoligonucleotides. PCR mutagenesis is also suitable for making variantsof the first or second polypeptide DNA. While the following discussionrefers to DNA, it is understood that the technique also findsapplication with RNA. The PCR technique generally refers to thefollowing procedure (see Erlich, Science, 252:1643 1650 (1991), thechapter by R. Higuchi, p. 61 70).

Additional modifications are also contemplated to further enhance thespecificity of interaction between the two heavy chains. Accordingly,the present invention incorporates a covalent link between the two heavychains (e.g. on the CH3 domains).

Examples of covalent links contemplated by the present invention includeamide links and disulfide links.

Thus, for example the present invention contemplates introduction of afree thiol which forms an intermolecular disulfide bond between the twoheavy chains of the antibody. The free thiol may be introduced into theinterface of one of the heavy chains by substituting a naturallyoccurring residue of the heavy chain with, for example, a cysteine at aposition allowing for the formation of a disulfide bond between theheavy chains.

The phrase “free thiol-containing compound” as used herein refers to acompound that can be incorporated into or reacted with an amino acid ofa polypeptide interface of the invention such that the free thiol moietyof the compound is positioned to interact with a free thiol of moiety atthe interface of additional polypeptide of the invention to form adisulfide bond. Preferably, the free thiol-containing compound iscysteine.

According to a specific embodiment, the first heavy chain comprises aS354C mutation (i.e. serine to cysteine); and the second heavy chaincomprises a Y349C mutation (tyrosine to cysteine).

As well as having modifications in their heavy chains, at least onelight chain of the antibodies described herein is also modified suchthat there is a first covalent link between a first heavy chain and afirst light chain and a second covalent link between a second heavychain and a second light chain, wherein a position of the first covalentlink relative to the first heavy chain is different to a position of thesecond covalent link relative to the second heavy chain.

The positioning of the first and second covalent link is selected suchthat pairing between a heavy chain with its cognate light chain isfacilitated, whilst the specificity and stability of the antibody is notreduced by more than 20% or preferably by more than 10% or even morepreferably by more than 5% as compared to the individual antibodies fromwhich it is generated.

According to another embodiment, the covalent link between the firstheavy chain to its cognate light chain is positioned between the C_(H1)and the C_(L) region and the covalent link between the second heavychain to its cognate light chain is positioned between the V_(H) and theV_(L) region.

Examples of covalent links contemplated by the present invention includefor example amide links, disulfide links and additional forms ofcovalent bonds occurring between site-specifically inserted amino acidresidues, including non-natural amino acids (see Wu, X., Schultz, P. G.“Synthesis at the Interface of Chemistry and Biology.” J. Am. Chem.Soc., 131(35):12497-515, 2009; Hutchins B M, Kazane S A, Staflin K,Forsyth J S, Felding-Habermann B, Schultz P G, Smider V V. Site-specificcoupling and sterically controlled formation of multimeric antibody fabfragments with unnatural amino acids J Mol Biol. 2011 Mar. 4;406(4):595-603. Epub 2011 Jan. 13; Liu C C, Schultz P G. Adding newchemistries to the genetic code. Annu Rev Biochem. 2010; 79:413-44.Review, all of which are incorporated herein by reference).

Accordingly, the present invention contemplates mutating at least one ofthe heavy chains and its cognate light chain such that at least onenaturally occurring (i.e. native) disulfide bond that connects the twomolecules can no longer be generated. Typically, this is effected bydeleting (or substituting) the cysteines at the positions describedherein above.

As used herein, the phrase “native disulfide bond” refers to theinterchain disulfide bond that connects a heavy chain to its cognatelight chain (typically between the constant region of the light chainand the CH1 region of the heavy chain) encoded in a naturally occurringgermline antibody gene.

Substitution of the cysteine is typically effected by replacing theamino acid with one similar in size and charge (i.e. a conservativeamino acid, such as cysteine to alanine).

The present invention contemplates that the first covalent link is anaturally occurring disulfide bond and the second covalent link is anon-naturally occurring covalent bond, (e.g. an engineered disulfidebond), wherein at least one cysteine amino acid residue has beeninserted into the chain—i.e. an engineered cysteine.

The term “engineered cysteine” as used herein, refers to a cysteinewhich has been introduced into the antibody fragment sequence at aposition where a cysteine does not occur in the natural germlineantibody sequence.

Alternatively, both the first and second covalent links may benon-naturally occurring and the cysteines (which in the non-modifiedantibody serve as amino acid residues to generate disulfide bonds) maybe replaced by other amino acids that are not capable of serving asamino acid residues to generate covalent bonds.

Information regarding the antibody of interest is required in order toproduce proper placement of the disulfide bond. The amino acid sequencesof the variable regions that are of interest are compared by alignmentwith those analogous sequences in the well-known publication by Kabatand Wu [Sequences of Proteins of Immunological Interest,” E. Kabat, etal., U.S. Government Printing Office, NIH Publication No. 91-3242(1990], incorporated herein by reference, to determine which sequencescan be mutated so that cysteine is encoded for in the proper position ofeach heavy and light chain variable region to provide a disulfide bondin the framework regions of the desired antibody.

After the sequences are aligned, the amino acid positions in thesequence of interest that align with the following positions in thenumbering system used by Kabat and Wu are identified: positions 43, 44,45, 46, and 47 (group 1) and positions 103, 104, 105, and 106 (group 2)of the heavy chain variable region; and positions 42, 43, 44, 45, and 46(group 3) and positions 98, 99, 100, and 101 (group 4) of the lightchain variable region. In some cases, some of these positions may bemissing, representing a gap in the alignment.

Then, the nucleic acid sequences encoding the amino acids at two ofthese identified positions are changed such that these two amino acidsare mutated to cysteine residues. Contemplated pairs of amino acids tobe selected are: V_(H)44-V_(L)100, V_(H)105-V_(L)43, V_(H)105-V_(L)42,V_(H)44-V_(L)101, V_(H)106-V_(L)43, V_(H)104-V_(L)43, V_(H)44-V_(L)99,V_(H)45-V_(L)98, V_(H)46-V_(L)98, V_(H)103-V_(L)43, V_(H)103-V_(L)44,V_(H)103-V_(L)45.

Most preferably, substitutions of cysteine are made at the positions:V_(H)44-V_(L)100; or V_(H)105-V_(L)43. (The notation V_(H)44-V_(L)100,for example, refers to a polypeptide with a V_(H) having a cysteine atposition 44 and a cysteine in V_(L) at position 100; the positions beingin accordance with the numbering given by Kabat and Wu.) Note that withthe assignment of positions according to Kabat and Wu, the numbering ofpositions refers to defined conserved residues and not to actualsequentially numbered amino acid positions in a given antibody. Forexample, CysL100 (of Kabat and Wu) which is used to generate ds(Fv)B3 asdescribed in the example below, actually corresponds to position 105 ofB3(V_(L)).

According to one embodiment, selection of which amino acid to mutate maybe effected according to the rules set out in U.S. Pat. No. 5,747,654,incorporated herein by reference. The sites of mutation to the cysteineresidues can be identified by review of either the actual antibody orthe model antibody of interest as exemplified below. Computer programsto create models of proteins such as antibodies are generally availableand well-known to those skilled in the art (see Kabat and Wu; Loew, etal., Int. J. Quant. Chem., Quant. Biol. Symp., 15:55-66 (1988);Bruccoleri, et al., Nature, 335:564-568 (1988); Chothia, et al.,Science, 233:755-758 (1986), all of which are incorporated herein byreference. Commercially available computer programs can be used todisplay these models on a computer monitor, to calculate the distancebetween atoms, and to estimate the likelihood of different amino acidsinteracting (see, Ferrin, et al., J. Mol. Graphics, 6:13-27 (1988),incorporated by reference herein). For example, computer models canpredict charged amino acid residues that are accessible and relevant inbinding and then conformationally restricted organic molecules can besynthesized. See, for example, Saragovi, et al., Science, 253:792(1991), incorporated by referenced herein. In other cases, anexperimentally determined actual structure of the antibody may beavailable.

According to one embodiment, a pair of suitable amino acid residuesshould (1) have a C_(α)—C_(α) distance between the two residues lessthan or equal to 8 ANG, preferably less than or equal to 6.5 ANG(determined from the crystal structure of antibodies which are availablesuch as those from the Brookhaven Protein Data Bank) and (2) be as faraway from the CDR region as possible. Once they are identified, they canbe substituted with cysteins.

Modifications of the genes to encode cysteine at the target point may bereadily accomplished by well-known techniques, such asoligonucleotides-directed mutagenesis (as described herein above),site-directed mutagenesis (see, Gillman and Smith, Gene, 8:81-97 (1979)and Roberts, S., et al, Nature, 328:731-734 (1987), both of which areincorporated herein by reference), by the method described in Kunkel,Proc. Natl. Acad. Sci. USA 82:488-492 (1985), incorporated by referenceherein, by total gene synthesis (Hughes, R. A. et al, Methods inEnzymology, Volume 498 p. 277-309 (2011)) or by any other means known inthe art.

Antibodies of some embodiments of the present invention may be from anymammalian origin including human, porcine, murine, bovine, goat, equine,canine, feline, ovine and the like. The antibody may be a heterologousantibody.

As used herein a “heterologous antibody” is defined in relation to atransgenic host such as a plant expressing the antibody.

According to some embodiments of the invention, the antibody is anisolated intact antibody (i.e., substantially free of cellular materialother antibodies having different antigenic specificities and/or otherchemicals).

As used herein “recombinant antibody” refers to intact antibodies thatare prepared, expressed, created or isolated by recombinant means, suchas (a) antibodies isolated from an animal (e.g., mouse) that istransgenic for immunoglobulin genes (e.g., human immunoglobulin genes)or hybridoma prepared therefrom; (b) antibodies isolated from a hostcell transformed to express the antibody; (c) antibodies isolated from arecombinant antibody library; and (d) antibodies prepared, expressed,created or isolated by any other means that involve splicing ofimmunoglobulin gene sequences to other DNA sequences. In certainembodiments immunoglobulin of the present invention may have variableand constant regions derived from human germline immunoglobulinsequences. In other embodiments, such recombinant human antibodies canbe subjected to in vitro mutagenesis and thus the amino acid sequencesof the V_(H) and V_(L) regions of the recombinant antibodies comprisesequences that while derived from and related to human germline V_(H)and V_(L) sequences, may not naturally exist within the human antibodygermline repertoire in vivo.

The following exemplary embodiments of antibodies are encompassed by thescope of the invention.

A used herein “human antibody” refers to intact antibodies havingvariable regions in which both the framework and CDR regions are derivedfrom human germline immunoglobulin sequences as described, for example,by Kabat et al. (see Kabat 1991, Sequences of proteins of immunologicalInterest, 5^(th) Ed. NIH Publication No. 91-3242). The constant regionof the human antibody is also described from human germlineimmunoglobulin sequences. The human antibodies may include aminoresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site directed mutagenesis in vitro orsomatic mutation in vivo). However, the term “human antibody”, as usedherein, is not intended to include antibodies in which CDR sequencesderived from the germline of another mammalian species, such as a mouse,have been grafted onto human framework sequences.

As used herein, a “chimeric antibody” refers to an intact antibody inwhich the variable regions derive from a first species and the constantregions are derived from a second species. Chimeric immunoglobulins canbe constructed by genetic engineering from immunoglobulin gene segmentsbelonging to different species (e.g., VH and VL domains from a mouseantibody with constant domains of human origin).

As used herein “humanized immunoglobulin” refers to an intact antibodyin which the minimum mouse part from a non-human (e.g., murine) antibodyis transplanted onto a human antibody; generally humanized antibodiesare 5-10% mouse and 90-95% human.

In general, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin consensus sequence. The humanized antibodyoptimally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin [Jones etal., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329(1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introduction of human immunoglobulinloci into transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar,Intern. Rev. Immunol. 13, 65-93 (1995).

The antibodies of the present invention may be conjugated to afunctional moiety such as a detectable or a therapeutic moiety.

Various types of detectable or reporter moieties may be conjugated tothe antibody of the invention. These include, but not are limited to, aradioactive isotope (such as ^([125])iodine), a phosphorescent chemical,a chemiluminescent chemical, a fluorescent chemical (fluorophore), anenzyme, a fluorescent polypeptide, an affinity tag, and molecules(contrast agents) detectable by Positron Emission Tomagraphy (PET) orMagnetic Resonance Imaging (MRI).

Examples of suitable fluorophores include, but are not limited to,phycoerythrin (PE), fluorescein isothiocyanate (FITC), Cy-chrome,rhodamine, green fluorescent protein (GFP), blue fluorescent protein(BFP), Texas red, PE-Cy5, and the like. For additional guidanceregarding fluorophore selection, methods of linking fluorophores tovarious types of molecules see Richard P. Haugland, “Molecular Probes:Handbook of Fluorescent Probes and Research Chemicals 1992-1994”, 5thed., Molecular Probes, Inc. (1994); U.S. Pat. No. 6,037,137 toOncoimmunin Inc.; Hermanson, “Bioconjugate Techniques”, Academic PressNew York, N.Y. (1995); Kay M. et al., 1995. Biochemistry 34:293; Stubbset al., 1996. Biochemistry 35:937; Gakamsky D. et al., “EvaluatingReceptor Stoichiometry by Fluorescence Resonance Energy Transfer,” in“Receptors: A Practical Approach,” 2nd ed., Stanford C. and Horton R.(eds.), Oxford University Press, U K. (2001); U.S. Pat. No. 6,350,466 toTargesome, Inc.]. Fluorescence detection methods which can be used todetect the antibody when conjugated to a fluorescent detectable moietyinclude, for example, fluorescence activated flow cytometry (FACS),immunofluorescence confocal microscopy, fluorescence in-situhybridization (FISH) and fluorescence resonance energy transfer (FRET).

Numerous types of enzymes may be attached to the antibody of theinvention [e.g., horseradish peroxidase (HRP), beta-galactosidase, andalkaline phosphatase (AP)] and detection of enzyme-conjugated antibodiescan be performed using ELISA (e.g., in solution), enzyme-linkedimmunohistochemical assay (e.g., in a fixed tissue), enzyme-linkedchemiluminescence assay (e.g., in an electrophoretically separatedprotein mixture) or other methods known in the art [see e.g., KhatkhatayM I. and Desai M., 1999. J Immunoassay 20:151-83; Wisdom G B., 1994.Methods Mol Biol. 32:433-40; Ishikawa E. et al., 1983. J Immunoassay4:209-327; Oellerich M., 1980. J Clin Chem Clin Biochem. 18:197-208;Schuurs A H. and van Weemen B K., 1980. J Immunoassay 1:229-49).

The affinity tag (or a member of a binding pair) can be an antigenidentifiable by a corresponding antibody [e.g., digoxigenin (DIG) whichis identified by an anti-DIG antibody) or a molecule having a highaffinity towards the tag [e.g., streptavidin and biotin]. The antibodyor the molecule which binds the affinity tag can be fluorescentlylabeled or conjugated to enzyme as described above.

Various methods, widely practiced in the art, may be employed to attacha streptavidin or biotin molecule to the antibody of the invention. Forexample, a biotin molecule may be attached to the antibody of theinvention via the recognition sequence of a biotin protein ligase (e.g.,BirA) as described in the Examples section which follows and inDenkberg, G. et al., 2000. Eur. J. Immunol. 30:3522-3532. Alternatively,a streptavidin molecule may be attached to an antibody fragment, such asa single chain Fv, essentially as described in Cloutier S M. et al.,2000. Molecular Immunology 37:1067-1077; Dubel S. et al., 1995. JImmunol Methods 178:201; Huston J S. et al., 1991. Methods in Enzymology203:46; Kipriyanov S M. et al., 1995. Hum Antibodies Hybridomas 6:93;Kipriyanov S M. et al., 1996. Protein Engineering 9:203; Pearce L A. etal., 1997. Biochem Molec Biol Intl 42:1179-1188).

Functional moieties, such as fluorophores, conjugated to streptavidinare commercially available from essentially all major suppliers ofimmunofluorescence flow cytometry reagents (for example, Pharmingen orBecton-Dickinson).

According to some embodiments of the invention, biotin conjugatedantibodies are bound to a streptavidin molecule to form a multivalentcomposition (e.g., a dimmer or tetramer form of the antibody).

Table 1 provides non-limiting examples of identifiable moieties whichcan be conjugated to the antibody of the invention.

TABLE 1 Nucleic Acid sequence Identifiable Amino Acid sequence (GenBankMoiety (GenBank Accession No.) Accession No.) Green Fluorescent AAL33912AF435427 protein Alkaline AAK73766 AY042185 phosphatase PeroxidaseCAA00083 A00740 Histidine tag Amino acids 264-269 of Nucleotides 790-807of GenBank Accession No. GenBank Accession No. AAK09208 AF329457 Myc tagAmino acids 273-283 of Nucleotides 817-849 of GenBank Accession No.GenBank Accession No. AAK09208 AF329457 Biotin lygase tag LHHILDAQ KMVWNHR/ orange AAL33917 AF435432 fluorescent protein Beta ACH42114EU626139 galactosidase Streptavidin AAM49066 AF283893 Table 1.

As mentioned, the antibody may be conjugated to a therapeutic moiety.The therapeutic moiety can be, for example, a cytotoxic moiety, a toxicmoiety, a cytokine moiety and a second antibody moiety comprising adifferent specificity to the antibodies of the invention.

Non-limiting examples of therapeutic moieties which can be conjugated tothe antibody of the invention are provided in Table 2, hereinbelow.

TABLE 2 Amino acid sequence Nucleic acid sequence Therapeutic (GenBankAccession (GenBank Accession moiety No.) No.) Pseudomonas ABU63124 - SEQID EU090068 - SEQ ID exotoxin NO: 42 NO: 51 Diphtheria AAV70486 - SEQ IDAY820132.1 - SEQ ID toxin NO: 43 NO: 52 interleukin 2 CAA00227 - SEQ IDA02159 - SEQ ID NO: 53 NO: 44 CD3 P07766 - SEQ ID NO: 45 X03884 - SEQ IDNO: 54 CD16 NP_000560.5 - SEQ ID NM_000569.6 - SEQ ID NO: 46 NO: 55interleukin 4 NP_000580.1 - SEQ ID NM_000589.2 - SEQ ID NO: 47 NO: 56HLA-A2 P01892 - SEQ ID NO: 48 K02883 - SEQ ID NO: 57 interleukin 10P22301 - SEQ ID NO: 49 M57627 - SEQ ID NO: 58 Ricin toxin EEF27734 - SEQID EQ975183 - SEQ ID NO: 50 NO: 59

The functional moiety may be conjugated to the V_(H) or the V_(L)sequence at either the N- or C-terminus or be inserted into otherprotein sequences in a suitable position. For example, for Pseudomonasexotoxin (PE) derived fusion proteins, either V_(H) or V_(L) should belinked to the N-terminus of the toxin or be inserted into domain III ofPE. For Diphtheria toxin-derived antibodies, V_(H) or V_(L) ispreferably linked to the C-terminus of the toxin.

It will be appreciated that such fusions can also be effected usingchemical conjugation (i.e., not by recombinant DNA technology).

The V_(H) and V_(L) sequences for application in this invention can beobtained from antibodies produced by any one of a variety of techniquesknown in the art.

Methods of producing polyclonal and monoclonal antibodies are well knownin the art (See for example, Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, New York, 1988, incorporatedherein by reference). Typically, antibodies are provided by immunizationof a non-human animal, preferably a mouse, with an immunogen comprisinga desired antigen or immunogen. Alternatively, antibodies may beprovided by selection of combinatorial libraries of immunoglobulins, asdisclosed for instance in Ward et al (Nature 341 (1989) 544). Thus anymethod of antibody production is envisaged according to the presentteachings as long as an immunoglobulin antibody is finally expressed inthe bacterial host.

The step of immunizing a non-human mammal with an antigen may be carriedout in any manner well known in the art for stimulating the productionof antibodies in a mouse (see, for example, E. Harlow and D. Lane,Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1988)). In a preferred embodiment, thenon-human animal is a mammal, such as a rodent (e.g., mouse, rat, etc.),bovine, porcine, horse, rabbit, goat, sheep, etc. As mentioned, thenon-human mammal may be genetically modified or engineered to produce“human” antibodies, such as the Xenomouse™ (Abgenix) or HuMAb-Mouse™(Medarex). Typically, the immunogen is suspended or dissolved in abuffer, optionally with an adjuvant, such as complete Freund's adjuvant.Methods for determining the amount of immunogen, types of buffers andamounts of adjuvant are well known to those of skill in the art and arenot limiting in any way on the present invention. These parameters maybe different for different immunogens, but are easily elucidated.

Similarly, the location and frequency of immunization sufficient tostimulate the production of antibodies is also well known in the art. Ina typical immunization protocol, the non-human animals are injectedintraperitoneally with antigen on day 1 and again about a week later.This is followed by recall injections of the antigen around day 20,optionally with adjuvant such as incomplete Freund's adjuvant. Therecall injections are performed intravenously or intraperitoneally andmay be repeated for several consecutive days. This is followed by abooster injection at day 40, either intravenously or intraperitoneally,typically without adjuvant. This protocol results in the production ofantigen-specific antibody-producing B cells after about 40 days. Otherprotocols may also be utilized as long as they result in the productionof B cells expressing an antibody directed to the antigen used inimmunization.

In an alternate embodiment, lymphocytes from a non-immunized non-humanmammal are isolated, grown in vitro, and then exposed to the immunogenin cell culture. The lymphocytes are then harvested and the fusion stepdescribed below is carried out.

For monoclonal antibodies, the next step is the isolation of splenocytesfrom the immunized non-human mammal and the subsequent fusion of thosesplenocytes with an immortalized cell in order to form anantibody-producing hybridoma. The isolation of splenocytes from anon-human mammal is well-known in the art and typically involvesremoving the spleen from an anesthetized non-human mammal, cutting itinto small pieces and squeezing the splenocytes from the splenic capsuleand through a nylon mesh of a cell strainer into an appropriate bufferso as to produce a single cell suspension. The cells are washed,centrifuged and re-suspended in a buffer that lyses any red blood cells.The solution is again centrifuged and remaining lymphocytes in thepellet are finally re-suspended in fresh buffer.

Once isolated and present in single cell suspension, the lymphocytes arefused to an immortal cell line. This is typically a mouse myeloma cellline, although many other immortal cell lines useful for creatinghybridomas are known in the art. Preferred murine myeloma lines include,but are not limited to, those derived from MOPC-21 and MPC-11 mousetumors available from the Salk Institute Cell Distribution Center, SanDiego, Calif. U.S.A., X63 Ag8653 and SP-2 cells available from theAmerican Type Culture Collection, Rockville, Md. U.S.A. The fusion iseffected using polyethylene glycol or the like. The resulting hybridomasare then grown in selective media that contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells. For example, if the parental myeloma cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (HAT medium), which substances prevent thegrowth of HGPRT-deficient cells.

The hybridomas are typically grown on a feeder layer of macrophages. Themacrophages are preferably from littermates of the non-human mammal usedto isolate splenocytes and are typically primed with incomplete Freund'sadjuvant or the like several days before plating the hybridomas. Fusionmethods are described in (Goding, “Monoclonal Antibodies: Principles andPractice,” pp. 59-103 (Academic Press, 1986.

The cells are allowed to grow in the selection media for sufficient timefor colony formation and antibody production. This is usually between 7and 14 days. The hybridoma colonies are then assayed for the productionof antibodies that bind the immunogen/antigen. The assay is typically acolorimetric ELISA-type assay, although any assay may be employed thatcan be adapted to the wells that the hybridomas are grown in. Otherassays include immunoprecipitation and radioimmunoassay. The wellspositive for the desired antibody production are examined to determineif one or more distinct colonies are present. If more than one colony ispresent, the cells may be re-cloned and grown to ensure that only asingle cell has given rise to the colony producing the desired antibody.Positive wells with a single apparent colony are typically recloned andre-assayed to insure only one monoclonal antibody is being detected andproduced.

Hybridomas that are confirmed to be producing a monoclonal antibody arethen grown up in larger amounts in an appropriate medium, such as DMEMor RPMI-1640. Alternatively, the hybridoma cells can be grown in vivo asascites tumors in an animal.

After sufficient growth to produce the desired monoclonal antibody, thegrowth media containing monoclonal antibody (or the ascites fluid) isseparated away from the cells and the monoclonal antibody presenttherein is purified. Purification is typically achieved by gelelectrophoresis, dialysis, chromatography using protein A or proteinG-Sepharose, or an anti-mouse Ig linked to a solid support such asagarose or Sepharose beads (all described, for example, in the AntibodyPurification Handbook, Amersham Biosciences, publication No. 18-1037-46,Edition A C, the disclosure of which is hereby incorporated byreference). The bound antibody is typically eluted from protein A,protein G or protein L columns by using low pH buffers (glycine oracetate buffers of pH 3.0 or less) with immediate neutralization ofantibody-containing fractions. These fractions are pooled, dialyzed, andconcentrated as needed.

DNA encoding the heavy and light chains of the antibody may be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of antibodies such as murine orhuman). Once isolated, the DNA can be ligated into expression vectors,which are then transfected into host cells.

The antibodies according to the invention are typically produced byrecombinant means.

The DNA sequences encoding the immunoglobulin light chain and heavychain polypeptides may be independently inserted into separaterecombinant vectors or one single vector, which may be any vector, whichmay conveniently be subjected to recombinant DNA procedures, and thechoice of vector will often depend on the host cell into which it is tobe introduced.

Methods for recombinant production are widely known in the state of theart and comprise protein expression in prokaryotic and eukaryotic cellswith subsequent isolation of the antibody and usually purification to apharmaceutically acceptable purity.

For the expression of the antibodies as aforementioned in a host cell,nucleic acids encoding the respective modified light and heavy chainsare inserted into expression vectors by standard methods.

The procedures used to ligate the DNA sequences coding for thepolypeptides, the promoter (e.g., constitutive or inducible) andoptionally the terminator sequence, respectively, and to insert theminto suitable vectors containing the information necessary forreplication, are well known to persons skilled in the art (see, forinstance, Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y., 1989).

Expression is performed in appropriate prokaryotic or eukaryotic hostcells like CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells,PER.C6 cells, yeast, or bacterial cells, and the antibody is recoveredfrom the cells (supernatant or cells after lysis).

The present invention contemplates expressing each component of theantibody in its own individual host cell, or various combinations of theantibody components in their own host cells. Thus for example, the lightchains may be expressed in one host cell and the heavy chains in anotherhost cell. Alternatively, one light chain and one heavy chain isexpressed in one host cell and the second light chain and the secondheavy chain is expressed in another host cell. Still alternatively, boththe heavy chains and both the light chains may be expressed in the samehost cell.

It will be appreciated that when both the heavy chains and both thelight chains are expressed in the same host cell, in vitro assembly ofthe chains is not necessary and only purification of the antibodies formthe conditioned medium i.e. by protein A chromatography is required (Seefor example: Jackman J, J Biol Chem. 2010 Jul. 2; 285(27):20850-9. Epub2010 May 5).

When at least one of the chains is expressed in a different host cell tothe other three chains, in vitro assembly of the chains is required.

According to a specific embodiment, the host cell comprises bacterialcells.

According to another embodiment the antibodies are generated asinclonals as described in WO2009/107129 incorporated herein byreference.

The bacterial host may be selected capable of producing the recombinantproteins (i.e., heavy and light chains) as inclusion bodies (i.e.,nuclear or cytoplasmic aggregates of stainable substances).

The host cells (e.g., first host cell and second host cell) used can beof identical species or different species.

According to specific embodiments of the present invention the hostcells are selected from a Gram-negative bacterium/bacteria.

As used herein “Gram negative bacteria” refers to bacteria havingcharacteristic staining properties under the microscope, where theyeither do not stain or are decolorized by alcohol during Gram's methodof staining. Gram negative bacteria generally have the followingcharacteristics: (i) their cell wall comprises only a few layers ofpeptidoglycans (which is present in much higher levels in Gram positivebacteria); (ii) the cells are surrounded by an outer membrane containinglipopolysaccharide (which consists of Lipid A, core polysaccharide, and0-polysaccharide) outside the peptideglycan layer; (iii) porins exist inthe outer membrane, which act like pores for particular molecules; (iv)there is a space between the layers of peptidoglycan and the secondarycell membrane called the periplasmic space; (v) the S-layer is directlyattached to the outer membrane, rather than the peptidoglycan (vi)lipoproteins are attached to the polysaccharide backbone, whereas inGram positive bacteria no lipoproteins are present.

Examples of Gram-negative bacteria which can be used in accordance withthe present teachings include, but are not limited to, Escherichia coliPseudomonas, erwinia and Serratia. It should be noted that the use ofsuch Gram-negative bacteria other than E. coli such as Pseudomonas as ahost cell would provide great economic value owing to both the metabolicand physiologic properties of pseudomonas. Under certain conditions,pseudomonas, for example, can be grown to higher cell culture densitiesthan E. coli thus providing potentially greater product yields.

Examples of bacterial expression vectors suitable for use in accordancewith the present teachings include, but are not limited to, pET™systems, the T7 systems and the pBAD™ system, which are well known inthe art.

Methods of introducing expression vectors into bacterial host cells arewell known in the art and mainly depend on the host system used.

The host cells can either be co-cultured in the same medium, or culturedseparately.

Host cells are cultured under effective conditions, which allow for theexpression of high amounts of recombinant heavy and light chain.Effective culture conditions include, but are not limited to, effectivemedia, bioreactor, temperature, pH and oxygen conditions that permitrecombinant protein production. An effective medium refers to any mediumin which a bacterium is cultured to produce the recombinant protein ofthe present invention. Such a medium typically includes an aqueoussolution having assimilable carbon, nitrogen and phosphate sources, andappropriate salts, minerals, metals and other nutrients, such asvitamins. Bacterial hosts of the present invention can be cultured inconventional fermentation bioreactors, shake flasks, test tubes,microtiter dishes, and petri plates, dependent on the desired amount.Culturing can be carried out at a temperature, pH and oxygen contentappropriate for a recombinant host. Such culturing conditions are withinthe expertise of one of ordinary skill in the art.

Once appropriate expression levels of immunoglobulin heavy and lightchains are obtained, the polypeptides are recovered from the inclusionbodies. Methods of recovering recombinant proteins from bacterialinclusion bodies are well known in the art and typically involve celllysis followed by solubilization in denaturant [e.g., De Bernardez-Clarkand Georgiou, “Inclusion bodies and recovery of proteins from theaggregated state” Protein Refolding Chapter 1:1-20 (1991). See alsoExamples section which follows, under “Expression of Inclonals in E.coli”].

Briefly, the inclusion bodies can be separated from the bulk ofcytoplasmic proteins by simple centrifugation giving an effectivepurification strategy. They can then be solubilized by strong denaturingagents like urea (e.g., 8 M) or guanidinium hydrochloride and sometimeswith extremes of pH or temperature. The denaturant concentration, timeand temperature of exposure should be standardized for each protein.Before complete solubilization, inclusion bodies can be washed withdiluted solutions of denaturant and detergent to remove some of thecontaminating proteins.

Finally, the solubilized inclusion bodies can be directly subjected tofurther purification through chromatographic techniques under denaturingconditions or the heavy and light chains may be refolded to nativeconformation before purification.

Thus, further purification of the reconstituted/refolded heavy and lightchain polypeptides (i.e., solubilized reduced polypeptides) can beeffected prior to, and alternatively or additionally, followingrefolding.

Methods of antibody purification are well known in the art and aredescribed hereinabove and in the Examples section which follows. Othermethods for purification of IgG are described in “Purification of IgGand insulin on supports grafted by sialic acid developing“thiophilic-like” interactions Hamid Lakhiaria and Daniel Mullerb,Journal of Chromatography B Volume 818, Issue 1, 15 Apr. 2005, Pages53-59.

Alternatively or additionally, purification can be affinity-basedthrough the identifiable or therapeutic moiety (e.g., using affinitycolumns which bind PE38 to purify antibodies that are fused to PE38).

Further purification of antibodies may be performed in order toeliminate cellular components or other contaminants, e.g. other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis, and others well known in the art. See Ausubel, F., etal., ed. Current Protocols in Molecular Biology, Greene Publishing andWiley Interscience, New York (1987). Different methods are wellestablished and widespread used for protein purification, such asaffinity chromatography with microbial proteins (e.g. protein A orprotein G affinity chromatography), ion exchange chromatography (e.g.cation exchange (carboxymethyl resins), anion exchange (amino ethylresins) and mixed-mode exchange), thiophilic adsorption (e.g. withbeta-mercaptoethanol and other SH ligands), hydrophobic interaction oraromatic adsorption chromatography (e.g. with phenyl-sepharose,aza-arenophilic resins, or m-aminophenylboronic acid), metal chelateaffinity chromatography (e.g. with Ni(II)- and Cu(II)-affinitymaterial), size exclusion chromatography, and electrophoretical methods(such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi,M. A., Appl. Biochem. Biotech. 75 (1998) 93-102).

To improve the refolding yield, the reconstituted heavy chains andreconstituted light chains are provided at a ratio selected to maximizethe formation of an intact antibody. To this end, a heavy to light chainmolar ratio of about 1:1 to 1:3, 1:1.5 to 1:3, 1:2 to 1:3 is. In anexemplary embodiment the heavy to light chain molar ratio is about 1:1.

When desired the immunoglobulin may be subjected to directed in vitroglycosylation, which can be done according to the method described byIsabelle Meynial-salles and Didier Combes. In vitro glycosylation ofproteins: An enzymatic approach. Journal of Biotechnology Volume 46,Issue 1, 18 Apr. 1996, Pages 1-14.

One aspect of the invention is a pharmaceutical composition comprisingan antibody according to the invention. Another aspect of the inventionis the use of an antibody according to the invention for the manufactureof a pharmaceutical composition. A further aspect of the invention is amethod for the manufacture of a pharmaceutical composition comprising anantibody according to the invention. In another aspect, the presentinvention provides a composition, e.g. a pharmaceutical composition,containing an antibody according to the present invention, formulatedtogether with a pharmaceutical carrier.

Antibodies and compositions (e.g., pharmaceutical composition)comprising same may be used in diagnostic and therapeutic applicationsand as such may be included in therapeutic or diagnostic kits.

Thus, compositions of the present invention may, if desired, bepresented in a pack or dispenser device, such as an FDA approved kit,which may contain one or more unit dosage forms containing the activeingredient i.e., antibody. The pack may, for example, comprise metal orplastic foil, such as a blister pack. The pack or dispenser device maybe accompanied by instructions for administration. The pack or dispensermay also be accommodated by a notice associated with the container in aform prescribed by a governmental agency regulating the manufacture, useor sale of pharmaceuticals, which notice is reflective of approval bythe agency of the form of the compositions or human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert. Compositions comprising a preparation of theinvention formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition, as is further detailed above.

One use of the antibody according to the invention is for the treatmentof diseases related to inflammation and infections.

As used herein the term “inflammation” refers to any medical conditionwhich comprises an inflammatory response in which migration of cells(e.g. to the lymph nodes) contributes to inflammation onset orprogression.

A number of diseases and conditions, which involve an inflammatoryresponse, can be treated using the methodology described hereinaboveincluding both chronic inflammatory diseases and acute inflammatorydiseases.

Examples of such diseases include inflammatory diseases associated withhypersensitivity.

Examples of hypersensitivity include, but are not limited to, Type Ihypersensitivity, Type II hypersensitivity, Type III hypersensitivity,Type IV hypersensitivity, immediate hypersensitivity, antibody mediatedhypersensitivity, immune complex mediated hypersensitivity, T lymphocytemediated hypersensitivity and DTH.

Other types of inflammatory diseases which may be treated with thebifunctional antibodies disclosed herein are autoimmune diseases,infectious diseases, graft rejection diseases, allergic diseases andcancerous diseases.

The term “cancer” as used herein refers to proliferative diseasesincluding by not limited to carcinoma, lymphoma, blastoma, sarcoma, andleukemia. Particular examples of cancerous diseases but are not limitedto: Myeloid leukemia such as Chronic myelogenous leukemia. Acutemyelogenous leukemia with maturation. Acute promyelocytic leukemia,Acute nonlymphocytic leukemia with increased basophils, Acute monocyticleukemia. Acute myelomonocytic leukemia with eosinophilia; Malignantlymphoma, such as Birkitt's Non-Hodgkin's; Lymphocytic leukemia, such asAcute lymphoblastic leukemia. Chronic lymphocytic leukemia;Myeloproliferative diseases, such as Solid tumors Benign Meningioma,Mixed tumors of salivary gland, Colonic adenomas; Adenocarcinomas, suchas Small cell lung cancer, Kidney, Uterus, Prostate, Bladder, Ovary,Colon, Sarcomas, Liposarcoma, myxoid, Synovial sarcoma, Rhabdomyosarcoma(alveolar), Extraskeletel myxoid chonodrosarcoma, Ewing's tumor; otherinclude Testicular and ovarian dysgerminoma, Retinoblastoma, Wilms'tumor, Neuroblastoma, Malignant melanoma, Mesothelioma, breast, skin,prostate, and ovarian.

Treatment of diseases may be effected by administering the antibodyalone, or together with a carrier as a pharmaceutical composition.

As used herein, “pharmaceutical carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g. by injection or infusion).

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. To administer a compound of the invention bycertain routes of administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation. For example, the compound may be administered to asubject in an appropriate carrier, for example, liposomes, or a diluent.

Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Pharmaceutical carriers include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intra-arterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol, sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions.

In addition, prolonged absorption of the injectable pharmaceutical formmay be brought about by the inclusion of agents which delay absorptionsuch as aluminum monostearate and gelatin.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carrierpreferably is an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by use of coating suchas lecithin, by maintenance of required particle size in the case ofdispersion and by use of surfactants. In many cases, it is preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol or sorbitol, and sodium chloride in the composition.

Other contemplated uses of the bispecific antibodies of the presentinvention include purification of analytes; in immunohistochemistry andenzyme immunoassays; for radioimaging and radioimmunotherapy and fordrug delivery.

Other contemplated uses are set forth in Cao Y, Suresh M R. Bispecificantibodies as novel bioconjugates. Bioconjug Chem. 1998November-December; 9(6):635-44, incorporated herein by reference.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Materials and Methods for Examples 1-4

Construction of Expression Vectors for Heavy and Light Chains:

The vector for production of antibody heavy and light chains in E. coliwere constructed on the backbone of pHAK vectors (Hakim and Benhar,2009). The heavy chains vectors were modified at the CH2-CH3 constantregions using Kunkel mutagenesis (Kunkel, 1985) to contain heavy-heavyheterodimer-preferable mutations according to “knobs-into-holes”approach (Merchant et al., 1998). To that end, DNA of the pHAK-IgHvector was prepared in E. coli CJ236 strain, infected with M13KO7 helperphage and released single-stranded uracil-containing plasmid DNA wascollected the next day using phenol-chloroform purification. The DNAsamples were incubated with either primer 1 (for introduction of “knob”mutations) or the mixture of primer 2, primer 3 and primer 4 (forintroduction of “hole” mutations) (Table 3, herein below) in TM buffer(0.01 M MgCl₂, 0.05 M Tris pH 7.5). In the next step, the DNA sampleswere incubated in presence of T7 polymerase and T4 ligase enzymes(supplied by 0.4 mM ATP, 0.4 mM dNTPs, 6 mM DTT) and transformed intoDH5a E. coli bacteria. The resulted constructs were named pHAK-HC-knob(carrying mutations T366W, S354C) and pHAK-HC-hole (carrying mutationsT366S, L368A, Y407V, Y349C). The mutation-containing regions weresubcloned using NsiI-NdeI restriction enzymes into pHAK-IgH-PE38 vector(Hakim and Benhar, 2009) that resulted in pHAK-HC-knob-PE38 andpHAK-HC-hole-PE38 vectors. The above constructs provided expression ofantibody heavy chain fused to PE38 toxin.

TABLE 3 Primer name Sequence 5′ to 3′ Notes Primer1GAAGCCTTTGACCAGGCAccaCAG Reverse primer for Kunkel S→C:GCTGACCTGGTTCTTGGTCATCTC mutagenesis that provides S354C T→WCTCCCGGcATGGGGGCAGGGTGT and T366W replacements on ACAC - SEQ ID NO: 1pHAK-IgH vector. Primer2 GGATGGGGGCAGGGTGcACACCTReverse primer for Kunkel Y→C GTGGTTCTCGG - SEQ ID NO: 2mutagenesis that provides Y349C replacement on pHAK-IgH vector. Primer3GGATAGAAGCCTTTGACCgcGC Reverse primer for Kunkel T→S:AGcTCAGGCTGACCTGGTTCTTG - mutagenesis that provides L368A L→ASEQ ID NO: 3 and T366S replacements on pHAK- IgH vector. Primer4GTCCACGGTGAGCTTGCTAacG Reverse primer for Kunkel Y→V AGGAAGAAGGAGCCGTC -mutagenesis that provides Y407 SEQ ID NO: 4replacement on pHAK-IgH vector. Primer5 ATATACATATGGACATTGTGCTGForward primer for PCR       NdeI -  amplification of variable region SEQ ID NO: 5 of light chain on pHAK-T427-IgL vector Primer6tatatacgtacgTTTGATTTCCAGTTTGG Reverse primer for A104C        BstWIreplacement in variable domain  TGCCgcaACCGAACGTCCGAGG - of T427-IgL.SEQ ID NO: 6 Primer7 tatataGAATTCTTACTCTCCCCTGTTReverse primer for removal of        EcoRIC218 amino acid codon from light GAAGCTCTTTGTG - chain sequence.SEQ ID NO: 7 Primer8 AAACAGAGGCCTGGACAGtGTC Forward primer for G44C       StuI replacement in variable domain  TGGAATGGATTG - of T427-IgH.SEQ ID NO: 8 Primer9 tatataGCTAGCGGAGGAGACTGTG Reverse primer for PCR       NheI amplification of variable region  AG -of heavy chain on pHAK-T427-IgH SEQ ID NO: 9 vector. Primer10GCCCAAATCTgccGACAAAACTCA Forward primer for C222A CACATGCCCACC -replacement in heavy chain SEQ ID NO: 10 constant region on pHAK-IgHvector. Primer11 TGTGTGAGTTTTGTCggcAGATTTG Reverse primer for C222AGGCTCAACTCTCTTG - replacement in heavy chain SEQ ID NO: 11constant region on pHAK-IgH vector. Primer12 GAGGAGATGACCAAGAACCAGGT -Reverse primer for amplification  SEQ ID NO: 12of heavy chain constant region  of pHAK-IgH vector. Primer13atataCATATGCAGGTCAAACTGC Forward primer for amplification        NdeI -of heavy chain variable region  SEQ ID NO: 13 of pHAK-T427-IgH vector.

To provide for efficient pairing of the heavy-light chains, the nativeinterchain di-sulfide bond was replaced with an engineered bond at analternative position in one IgH/IgL pair. The mutations that wereinserted in pHAK-LC-Cys were A104C in V_(L), and a C218del in C-Kappa.The mutations that were inserted in pHAK-HC-Cys were A44C in V_(H) andC222A in CH1. The construction of pHAK-LC-Cys vector included twosequential cloning steps. First, the light chain variable domain of theselected antibody was amplified with primer 5 and primer 6, digestedwith NdeI-BsiWI restriction enzymes and cloned to pHAK-IgL previouslydigested with the same enzymes. The resulted vector served as a templatefor amplification of IgL with primer 5 and primer 7, which was digestedwith NdeI-EcoRI enzymes and cloned to pHAK-IgL (NdeI-EcoRI digested). Inorder to construct pHAK-HC-Cys (A44C), the heavy chain variable regionof selected antibody was amplified with primer 8 and primer 9, followingStuI-NheI digestion and cloning into pHAK-IgH vector. The insertion ofthe C222A mutations into CH1 was carried out by amplification of two ofthe two PCR fragments that were generated by either 10 and 12 primers or11 and 13 primers, followed by assembly PCR with primer 12 and primer13. The assembled DNA fragment was digested with NdeI and BsrGIrestriction enzymes and cloned into previously constructed pHAK-HC-Cys(A44C) vector.

The combined pHAK-HC-Cys-knob vector (A44C, C222A, T366W, and S354C) wasconstructed by insertion of NdeI-SacII digested region of pHAK-HC-Cys topHAK-HC-knob vector. The light or heavy variable regions of desiredantibody were cloned on either pHAK-LC-based vector (using NdeI-BsiWIsubcloning) or pHAK-HC-based vector (using NdeI-NheI subcloning).

IgG Production in E. coli:

Heavy and light chains constructs based on pHAK-IgH and pHAK-IgL,respectively, were expressed in separate E. coli BL21 (DE3) pUBS500bacterial cultures as inclusion bodies. The inclusion bodies werepurified, denatured, mixed and refolded according to the Inclonals IgGproduction method (Hakim and Benhar, 2009). For bispecific IgGproduction the complement heavy chains were added at 1:1 molar ratio.The same rule was applied for the light chains.

Protein A Purification:

Following the refolding process IgG and IgG-based fusion proteins wereloaded on a protein A affinity column and separated from bacterialcontaminants and not efficiently refolded proteins. The proteins wereeluted with 0.1 mM citric acid neutralized with 1M Tris (HCl) pH 8.5followed by dialysis against 20 mM phosphate buffer solution (PBS) pH7.4. The protein final concentration was determined by absorbance at 280nm.

Gel Filtration Chromatography:

Gel filtration analysis was carried out on Amersham Pharmacia ÄKTA FPLCSystem to determine the molecular mass of the purified antibodies. Theprotein A purified proteins were applied to a Superdex 200 column,previously equilibrated with PBS (pH 7.4), and separated using the samebuffer at a flow rate of 0.5 ml/min. The molecular weight of examinedIgG-like proteins was determined by comparing its elution volume withthat of standard IgG (150 kDa) and IgG-based immunotoxin IgG-PE38 (225kDa).

SDS-PAGE Analysis:

Polyacrylamide gel electrophoresis of proteins was performed accordingto Laemmli (Laemmli, 1970) ⅕ volume of 5× sample buffer was added to theprotein samples followed by boiling for 5 min prior to the loading ontothe gel. 7.5%, 10% and 12% mini-gels were run at 120 V. For evaluationof full length IgG, the non-reduced samples (without β-mercaptoethanol)were loaded, while the reduced protein samples separated into heavy andlight chains components. Gels were stained with Coomassie blue solution(0.05% Coomassie R-250, 20% ethanol, 10% glacial acetic acid) for 2hours and washed in destain solution (20% ethanol, 10% glacial aceticacid) until protein bands could be clearly seen. The protein banddensity was analyzed by ImageMaster 1D scanning laser densitometer(Pharmacia, Sweden). Gels that were stained were loaded with 20 μg ofprotein per lane for non-purified fraction or 3-5 μg for purifiedproteins. Gels that were further processed by immunoblotting were loadedwith 1/10 that quantity.

Western Blot Analysis:

Proteins resolved by SDS-PAGE were electro-transferred onto thenitrocellulose membrane according to (Towbin et al., 1992). The membranewas blocked for at least 1 hour with PBS containing 5% non-fat milkpowder at room temperature with slow agitation. The membrane was washedwith PBS followed by incubation HRP conjugated goat-anti-human secondaryantibodies (Jackson Immunoresearch Laboratories, West Grove, Pa.). Afterthree washes with PBS containing 0.05% Tween-20 (PBST) and one wash withPBS the nitrocellulose filter was developed with the SuperSignal WestPico Chemiluminescent Substrate (Thermo Scientific, USA) as described bythe vendor.

ELISA Analysis:

The antigen binding by mono- and bispecific IgGs was determined asfollows: the 96-well ELISA plate was coated with 5 μg/ml of pure antigenin PBS 100 μl/well for overnight at 4° C. and blocked with 3% skim milk(in PBS) for 1 hour at 37° C. All subsequent steps were carried out atroom temperature (25° C.). Protein A purified proteins were applied ontothe plates in a three-fold dilution series in PBST for 1 hour incubationand washed with PBST for three times. Following the 1 hour incubationwith HRP conjugated secondary antibodies (1:5000 dilution in PBST, 100μl/well), the plates were washed in PBST and developed using chromogenicHRP substrate TMB and colour development was terminated with 1M H₂SO₄.The plated were read at 450 nm.

Example 1 Production of Full-Length IgG in E. coli Using InclonalsMethod

The Inclonals method for production of full-length IgG in E. colibacteria (Hakim and Benhar, 2009) includes using pHAK-IgH and pHAK-IgLvectors for production of IgH and IgL, respectively in separatebacterial cultures. The variable regions of heavy and light chainsdefine the antibody specificity while the constant region is common foreach vector. The protein expression, purification and refolding wascarried out according to the Inclonals protocol and the purifiedproteins were evaluated using SDS-PAGE, Western blot, size exclusionchromatography and antigen binding analysis. As opposed to mono-specificantibody, the bispecific IgGs consists of 2 different heavy chains and 2different light chains, thus expression and refolding steps includeconcomitant work with 4 proteins.

Example 2 Construction and Evaluation of Heavy-Heavy Chain Heterodimers

The “knobs-into-holes” approach (Ridgway et al., 1996) was implementedas a solution to preferable heterodimerization of different heavy chainsfor bispecific IgG production in E. coli. It was previously demonstratedthat introduction of 4 mutations (T366W in “knob” heavy chain and T366S,L368A, Y407V in “hole” heavy chain) and the asymmetric disulfide bond(S354C and Y349C on complement heavy chains) provided high (>95%)heterodimerization level of heavy chains in IgG produced in mammaliancells (Merchant et al., 1998), (FIG. 1A). The above mutations were usedfor construction of pHAK-HC-knob and pHAK-HC-hole vectors that were usedfor expression and examination of heavy chains, while the commonunmodified light chain served for all IgG constructs (FIG. 2A-H). T427(anti-CD30) and FRP5 (anti-erbB2) antibodies were used as model IgGs formethod evaluation (Harwerth et al., 1992; Nagata et al., 2004). Theantibody heavy and light chains were expressed as inclusion bodies,purified by centrifugation and analyzed by SDS-PAGE (FIG. 3). Therefolding of 4 antibody chains together followed by protein Apurification according to Inclonals protocol enabled production offull-length IgG.

For detailed characterization of heterodimerization yield the“hole-heavy” chain was expressed as fusion protein with PE38 toxin(Kreitman et al., 1992) that provided additional 38 kDa to proteinmolecular weight. As illustrated in FIGS. 4A-B, using SDS-PAGE analysisit was possible to distinguish between the homodimer of “knob” heavychains (150 kDa), the homodimer of “hole” toxin-fused heavy chains (230kDa) and the heterodimer of two different heavy chains (190 kDa). FIG.4B demonstrates that Inclonals' produced T427 “knobs-into-holes”antibody migrated as 190 kDa band on a non-reducing polyacrylamide geland could be separated to 3 components (IgL, IgH and IgH-PE38) underreducing conditions.

The attempt to produce “knob-knob” and “hole-hole” versions of IgG bysupplying the refolding solution with only one heavy chain type (either“knob” or “hole”) resulted in assembly failure of IgG and performance ofpartial-sized molecules (FIGS. 5A-B).

The evaluation of bispecific inclonals “knobs-into-holes” antibodiesusing size-exclusion chromatography demonstrated that protein majoritymigrated as a 190 kDa molecules while only small protein fractionrepresented homodimers (FIG. 6). Density analysis SDS-PAGE of theInclonals “knobs-into-holes” antibody concluded that >90% of E. coliproduced IgGs underwent heavy chains heterodimerization (FIGS. 7A-C).

In order to evaluate the binding activity of bispecific molecules the“knobs-into-holes” bispecific T427-FRP5 antibody was constructed. ThisIgG consisted of 4 different chains: FRP5-knob and T427-hole-PE38 heavychains, and FRP5 wt and T427 wt light chains. The PE38 toxin in thisconstruct was used as a detection signal for T427 heavy chain presence.The mono-specific T427 and FRP5 IgGs served as controls. Using indirectELISA the present inventors demonstrated the antibodies' binding abilityto each one of its antigens (erbB2 for FRP5 (FIG. 8A) and CD30 for T427(not shown)). The special ELISA (FIG. 8B) analysis examines the antibodybinding to FRP5 antigen while T427-PE38 chain was detected. This assaydemonstrated the presence of T427-FRP5 heterodimer that was able to bindits' two antigens.

Example 3 Construction and Evaluation of Heavy-Light Chains SpecificPairing

In order to introduce the disulfide bond between the two variabledomains to replace the native heavy-light interchain S—S bond, the T427antibody was used. This antibody has been extensively studied and its'cysteine positions for dsFv have been well defined (Nagata et al.,2004). Vectors pHAK-HC-Cys and pHAK-LC-Cys were constructed byreplacement of conventional cysteine position by dsFv defined. Theproduction of dsFv-like modified mono-specific IgG demonstrated theefficient formation of full length IgG stabilized by a single dsFv-likeheavy-light interchain S—S bond (FIG. 9, lane 3).

The construction of pHAK-HC-Cys-knob enabled the production of fullybispecific full length T427-FRP5 IgG (FIG. 9, lane 4).Heterodimerization of heavy chains was provided by “knobs-into-holes”strategy and heavy-light pair matching was ensured by asymmetricinterchain disulfide bond. Further, the IgG refolding solutions providedwith unpaired heavy and light chains did not generate complete IgGmolecules (FIG. 10).

Example 4

Two additional bispecific antibodies were produced, purified andevaluated, as described above in the materials and methods.

-   -   1. T427-αSA IgG: binding to CD30 and streptavidin (SA). The        bispecific antibody consisted of 4 chains: IgL-T427-Cys        (Cys104:Cys218del), IgH-T427-knob-Cys        (Cys44:Cys222Ala+S354C:T366W), IgL-αSA and IgH-αSA-hole        (Y349C:T366S:L368A:Y407V).    -   2. T427-αPE (B11 clone) IgG: binding to CD30 and PE38        (Pseudomonas exotoxin 38 kDa fragment). The bispecific antibody        consisted of 4 chains: IgL-T427-Cys (Cys104:Cys218del),        IgH-T427-knob-Cys (Cys44:Cys222Ala+S354C:T366W), IgL-αPE and        IgH-αPE-hole (Y349C:T366S:L368A:Y407V).

The anti-streptavidin (αSA) and anti-PE B11 clone (αPE) antibodies wereisolated as scFvs by affinity selecting the “Ronit1” antibody phagedisplay library (Azriel-Rosenfeld et al., 2004, J Mol Biol 335, 177-92).The heavy and light domains were cloned into the pHAK-IgH-hole andpHAK-IgL, respectively (as mentioned above). The chains were produced inE. coli bacteria as inclusion bodies, purified by centrifugation andanalyzed by SDS-PAGE electrophoresis (FIG. 11). The appropriate heavyand light chains were mixed, refolded and purified by Protein A affinitypurification for production of mono-specific T427, αSA, αPE andbispecific T427-αSA and T427-αPE antibodies according to Inclonalsprotocol (Hakim and Benhar, 2009). The antibodies were analyzed by ELISAfor binding activity to each antigen (FIGS. 12 and 13). As shown, thesebispecific antibodies were successfully produced and bound the twoantigens according to the specificities of the two arms. Specificity wasdemonstrated by negligible binding to bovine serum albumin (BSA).

Materials and Methods for Examples 5-9 Construction of pDual Vectors forExpression of IgGs in Mammalian Cells

The vector for production of antibody heavy and light chains in E. coliwere constructed on the backbone of pMAZ vectors (Mazor Y., et al. JImmunol Methods. 2007 Apr. 10; 321(1-2):41-59.). Two bi-cistronic pMAZvectors were constructed—pMAZ-IgH that carried the heavy chain and aNeomycin selection marker; and pMAZ-IgL that carried the light chain anda hygromycin selection marker. IgG expression was mediated byco-transfection of the two vectors, followed by double drug selectionfor obtaining stable transfectants.

The pDual vector was based on pMAZ-IgH vector that was previouslymutated using IgH-Apadel-NheI-For and IgH-BsrGI-Rev primers in order todelete the ApaI restriction site in the constant region. The next stepwas the construction of pCMV-IgL-term cassette and cloning it betweenthe KpnI-EcoRI restriction sites of pMAZ-IgH-Apadel vector. ThepCMV-IgL-term cassette was built by assembly of three PCR products thatincluded: 1) amplification of pCMV promoter using pCMV-KpnI-For andpCMV-Rev primers that provided the replacement of BssHI by ApaIrestriction site (this ApaI site will be unique in the plasmid since theApaI site that was present in the Fc coding region was mutated); 2)amplification of the T427 light chain antibody (VL+LC) using T427L-Forand T427L-Rev primers that provided the replacement of XbaI by NotIrestriction site; 3) amplification of the BGH polyadenylation site usingBGH-polyA-For and BGH-polyA-EcoRI-Rev primers. The above PCR productswere assembled into pCMV-IgL-term cassette by overlap-extensionpolymerase chain reaction (assembly PCR) followed by digestion with KpnIand EcoRI restriction enzymes and cloning into pMAZ-IgH-Apadel vector asdescribed above. The resulted vector was named pDual that was furtherused for cloning of variable domains of different antibodies usingApaI-BsiWI restriction sites for VL (kappa light chains, for lambdalight chains, a separate vector is required that carries a lambda lightchain, into which V-lambda variable domain should be cloned asApaI-AvrII restriction fragments) and BssHI-NheI restriction sites forVH.

A similar pDual vector was constructed that carries the hygromycinselection marker.

The list of primers used for generating the above described pDualvectors is summarized in Table 4, herein below.

TABLE 4 Primer name Sequence 5′ to 3′ Notes IgH-Apadel-tcctcaGCTAGCaccaagggAccatcggtcttccccctg Forward primer for NheI-For       NheI removal of ApaI SEQ ID NO: 60 restriction site at IgHconstant domain by silent mutation IgH-BsrGI- gcagggTGTACAcctgtggttcReverse primer for IgH- Rev        BsrGI ApadeI-NheI-For SEQ ID NO: 61pCMV- actgaaccttggagtcaGGTACCacattgat Forward primer for KpnI-For                  KpnI amplification of CMV Tattgagtagttattaatagpromoter SEQ ID NO: 62 pCMV-Rev GGGCCCctgtggagagaaaggcaaagtggatgReverse primer for  ApaI amplification of CMV SEQ ID NO: 63promoter and insertion of ApaI restriction site between ER secretionsignal and VL antibody region. T427L-ForctttgcctttctctccacagGGGCCCactccgac Forward primer for                     ApaI amplification of T427 attgtgctgacccaatcIgL and assembly with SEQ ID NO: 64 pCMV fragment. T427L-RevcggtttaaaaaacgggacctctggaGCGGCCGCtt Reverse primer for                           NotI amplification of T427attaacactctcccctgttgaagctctttgtg IgL that allows the SEQ ID NO: 65replacement of XbaI by NotI restriction site. BGH-polyA-tccagaggtcccgttttttaaaccggttttttaaaccgctg Forward primer for Foratcagcctcg amplification of SEQ ID NO: 66 polyadenylation site andassembly with T427 IgL fragment. BGH-polyA-tagctcgatccgtcgagaGAATTCccccagcat Reverse primer for EcoRI-Rev                   EcoRI amplification of gcctgctattgpolyadenylation site. SEQ ID NO: 67

Transfection of HEK293 T-REx™ Cells:

The calcium-phosphate transfection method was applied for introducing 1μg of the pDual or pMAZ plasmids into T-REx 293 cells, seeded 3×10⁵cells/well on 6-well plate 24 hours before transfection. For transienttransfection, the medium samples were collected 24, 48 and 72 hours posttransfection. In order to obtain the stable transfectants, the cellswere harvested 24 hours post transfection and seeded on DMEMsupplemented with appropriate antibiotics (1.2 mg/ml G418 and 0.2 mg/mlHygromycin). The stable clones were collected and their media wereevaluated for the presence of antibody.

IgG Production in HEK293 T-REx™ Cells:

The previously obtained stable clones were transferred to tissue cultureflasks (250 cm³) in DMEM supplement with 0.9 mg/ml G418 and 0.15 mg/mlHygromycin (75% of the regular concentration). The next day (or when thecells reached 80% confluence) the medium was changed to 50% DMEM(+L-Glu, PNS and bovine serum) and 50% DCCM1 (+L-Glu, PNS, serumfree)+75% of antibiotics concentration (0.9 mg/ml G418 and 0.15 mg/ml ofHygromycin) for 24 hours. The next day the medium was changed to 100%serum free DCCM1 (+L-Glu, PNS). The DCCM1 media from cells werecollected every 2-4 days and gently changed to new serum-free media. Itwas possible to collect up to 4 harvests from the flask.

Protein A Purification of IgG Produced in Mammalian Cells:

The collected DCCM1 medium from antibody secreting cells was centrifugedat 5500 rpm for 15 minutes and filtered using 0.45 μm filtrap. Themedium was diluted 1:20 with ×20 concentration phosphate buffer (400 mM)to final concentration of 20 mM Na₂HPO₄ and 20 mM NaH₂PO₄ and themixture was loaded onto protein A column at a flow rate of 1 ml/min. Theproteins were eluted with 0.1 mM citric acid (pH 3), neutralized with 1MTris (HCl) pH 8.5 which was followed by dialysis against 20 mM phosphatebuffer solution (PBS) pH 7.4. The protein final concentration wasdetermined by absorbance at 280 nm.

ELISA Analysis:

The antigen binding by mono- and bispecific IgGs was determined asfollows: 96-well ELISA plates were coated with 5 μg/ml of pure antigenin PBS 100 μl/well for overnight at 4° C. and blocked with 3% milk (inPBS) for 1 hr at 37° C. All subsequent steps were carried out at roomtemperature (25° C.). Protein-A purified proteins (or conditioned media)were applied onto the plates in a three-fold dilution series in PBST for1 hour incubation and washed with PBST for three times. Following the 1hour incubation with HRP-conjugated secondary antibody (1:5000 dilutionin PBST, 100 μl/well), the plates were washed in PBST and developedusing chromogenic HRP substrate TMB and colour development wasterminated with 1 M H₂SO₄. The plates were read at 450 nm.

Cell ELISA Analysis:

The A431/CD30 (expressing CD30, target antigen for T427) and SKBR3(expressing ErbB2, target antigen for FRP5) cell lines were maintainedin DMEM supplemented by 10% fetal calf serum, 1% L-glutamine and 1%penicillin-streptomycin and grown at 37° C. with 5% CO₂. The cells(2×10⁴/well) were seeded onto 96-well tissue culture plates in 100 μlmedium and grown at 37° C. for overnight. Following the overnight growththe medium was gently poured out and the cells were fixed with 3%glutaraldehyde solution in water for 15 minutes at room temperature. Thecells were washed with PBS and blocked with 5% BSA in PBS for 2 hours at37° C. All subsequent steps were carried out according the regular ELISAprotocol at room temperature (25° C.).

Dot Blot Analysis:

The 100 μl samples of 72 hours post-transfection cell conditioned mediawere diluted in an equal volume of PBS and applied via a vacuum manifoldonto a nitrocellulose membrane filter using a dot-blot apparatus(Schleicher and Schuell, USA). After blocking the membranes with 3%(v/v) non-fat milk in PBS for 1 hour at 37° C., the membrane was washedbriefly with PBS followed by incubation with goat-anti-human HRPconjugated secondary antibody for 1 hour at room temperature. Afterthree washes with PBS the membrane was developed with the ECL reagent(Pierce, USA).

SDS-Page Analysis:

Polyacrylamide gel electrophoresis of proteins was performed accordingto Laemmli (Laemmli, 1970) ⅕ volume of 5× sample buffer was added to theprotein samples followed by boiling for 5 minutes prior to loading ontothe gel. 7.5%, 10% and 12% mini-gels were run at 120 V. For evaluationof full length IgG, non-reduced samples (without β-mercaptoethanol) wereloaded, while the reduced protein samples separated into heavy and lightchains components. Gels were stained with Coomassie blue solution (0.05%Coomassie R-250, 20% ethanol, 10% glacial acetic acid) for 2 hours andwashed in destain solution (20% ethanol, 10% glacial acetic acid) untilprotein bands could be clearly seen. The protein band density wasanalyzed by ImageMaster 1D scanning laser densitometer (Pharmacia,Sweden). Gels that were stained were loaded with 20 μg of protein perlane for non-purified fraction or 3-5 μg for purified proteins. Gelsthat were further processed by immunoblotting were loaded with 1/10 thatquantity.

Western Blot Analysis:

Proteins resolved by SDS-PAGE were electro-transferred onto thenitrocellulose membrane according to (Towbin et al., 1992). The membranewas blocked for at least 1 hour with PBS containing 5% non-fat milkpowder at room temperature with slow agitation. The membrane was washedwith PBS followed by incubation HRP conjugated goat-anti-human secondaryantibodies (Jackson Laboratories, West Grove, Pa.). After three washeswith PBS containing 0.05% Tween-20 (PBST) and one wash with PBS thenitrocellulose filter was developed with the SuperSignal West PicoChemiluminescent Substrate (Thermo Scientific, USA) as described by thevendor.

Example 5 The Production of IgG in Mammalian Cells

The vector system used for the production of IgG in mammalian cells forproduction of bispecific antibodies was based on pMAZ vectors forproduction of monoclonal antibodies in mammalian cell culture (Mazor Yet al, 2007). Vector pMAZ-IgH was designed for human γ1 heavy chainexpression and pMAZ-IgL for human κ light chain expression. The variabledomains of light and heavy chains were introduced to the appropriatevector, co-transfected into HEK293 cells and stable antibody secretingclones were identified and kept. The starvation of cell clones to serumresulted in secretion of the desired antibody at a total yield of up to20 mg per liter of culture.

Example 6 The Construction of Dual Vector for Production of IgGMolecules in Mammalian Cells

The pDual vector was constructed by fusion of DNA fragments derived fromthe pMAZ-IgL and pMAZ-IgH vectors (Mazor Y et al, 2007) that werepreviously used for production of antibodies' light and heavy chainsindependently (FIG. 14), in order to build chimeric construct forproduction of light and heavy antibody chains using the same vector. TheIgL and IgH were constructed in pDual vector as separate cassettes underthe control of separate CMV promoters. The replacement of BssHI by ApaIrestriction site in the light chain cassette simplified the followingcloning of variable light and heavy domains into dual vector: ApaI-BsiWIwere used for cloning of VL and BssHI-NheI were used for cloning of VH.

Example 7 The Construction of Bispecific Vectors for Transfection toMammalian Cells

In order to construct the pDual-based vectors for production ofbispecific molecules, the “knob”, “hole” and “Cys” related mutationswere cloned from the pHAK vectors into the pDual system. The cloningprocess resulted in the series of constructs listed in Table 5. Thereplacement of Neo® with Hygro resistance cassette in pDual-T427 vectorswas carried out by subcloning of the cassette from pMAZ-IgL vector usingAvrII and KpnI restriction enzymes. The resulted pDual-Neo® andpDual-Hygro® vectors' pair can be used for transfection in mammaliancells and selection of stable clones that produce 4 different antibodychains: two heavy and two light chains within one cell line.

TABLE 5 Vector name Product pDual-T427 wt Neo^(R) IgL-T427 + IgH-T427pDual-FRP5 wt Neo^(R) IgL-FRP5 + IgH-FRP5 pDual-T427-L(wt)-H(knob)Neo^(R) IgL-T427 + IgH-T427(knob) pDual-FRP5-L(wt)-H(hole) Neo^(R)IgL-FRP5 + IgH-FRP5(hole) pDual-T427-L(Cys)-H(wt) Neo^(R)IgL-T427(Cys) + IgH-T427 pDual-T427-L(Cys)-H(knob) Neo^(R)IgL-T427(Cys) + IgH-T427(knob) pDual-T427-L(wt)-H(Cys-knob) IgL-T427 +IgH-T427(Cys-knob) Neo^(R) pDual-T427-L(Cys)-H(Cys-knob) IgL-T427(Cys) +Neo^(R) IgH-T427(Cys-knob) pDual-T427 wt Hygro^(R) IgL-T427 + IgH-T427pDual-T427-L(Cys)-H(Cys-knob) IgL-T427(Cys) + Hygro^(R)IgH-T427(Cys-knob)

Example 8 The Production and Evaluation of Bispecific IgG Molecules inTransient Transfected HEK293 T-REx™ Cells

The following example demonstrates the importance of the S—S bridgebetween the light and heavy chain of the antibody in IgG secretionsystem and proves that the “alternative Cysteine” theory for couplingthe appropriate light and heavy chain of bispecific antibody is relevantin mammalian production system as well as it had been demonstrated inthe E. coli produced bispecific “Inclonals”. The HEK293 T-REx™ cell linewas used for this study. The cells were transiently transfected witheither pDual-T427 wt (encoding wt IgG), pDual-T427-L(Cys)-H(wt)(encoding wt heavy chain and light chain that lacks the C-kappa cysteineand does contain the e engineered cysteine in VL, this should be a pairof chains that should not form an IgG) or with pMAZ-IgL+pMAZ-IgH(previous system for wt IgG production) pair as control. The evaluationof post-transfection medium by Western blot analysis showed that nofull-size antibodies were detected in media of pDual-T427-L(Cys)-H(wt)transfected cells, while the transfection of pDual wt construct producedthe detectable levels of the secreted antibody (up to 1 μg/ml incomparison to Erbitux dilutions) (FIGS. 15A and B).

The secretion of bispecific IgG molecules was also demonstrated and thepreference of bispecific IgG formation using “knobs-into-holes” and“alternative Cysteine” (also called “disulfide stabilization”)approaches was also estimated. The pDual vectors were transientlytransfected to HEK293 TRex cells and the secreted antibodies weredetected by Western blot analysis of conditioned media. The cells thatwere transfected with four pMAZ vectors (pMAZ-T427-IgL, pMAZ-T427-IgH,pMAZ-FRP5-IgL and pMAZ-FRP5-IgH) served as a control. The analysis ofexperiment demonstrated: 1) the “knobs-into-holes” approach is asolution for efficient heterodimerization of different heavy chains, 2)“alternative Cys” approach provide the solution for coupling of lightand heavy chains of the same antibody arm, 3) the combination of the twoabove approaches provides the secretion of full-length bispecific IgGantibodies in mammalian cells production systems. As shown in FIG. 16,when “wrong” combinations of chains are used, no full size IgG can beseen in the immunoblot analysis of conditioned media. Intact IgG can beobserved in cells that express monospecific IgG (lanes 1 and 2), incells that express two monospecific IgGs (lanes 5, two pDual vectors andlane 4 four pMAZ vectors) and in cells that express a bispecific IgG(lane 7).

Example 9 The Production and Evaluation Bispecific IgG Molecules inStable Transfected HEK293 T-REx™ Cells and Evaluation of Antigen Binding

In order to obtain stable antibody-secreting clones, the presentinventors co-transfected HEK293 T-REx™ cells withpDual-FRP5-L(wt)-H(hole) Neo® and pDual-T427-L(Cys)-H(Cys-knob) Hygro®vectors. The resultant Neo+Hygro resistant clones were verified fortheir ability: 1) to secrete antibody, 2) to bind both antigens, 3) tosecret full-length IgG for further purification. The preliminaryantibody secretion test was performed using Dot blot analysis (the testexample demonstrated in FIG. 17) and identified the low, medium and highsecreting clones. The clones marked as medium and high secretors wereexamined for their antigen binding activity. ELISA was carried out toevaluate the binding level of each clone to erbB2 (FRP5) and CD30 (T427)recombinant antibodies (FIG. 18). The several clones that were able tobind each of the antigens continued to the third step and were purifiedon protein A affinity column (FIG. 19). The purified antibodies wereanalyzed to determine their size, purity and binding activity to eitherrecombinant antigens or antigen-presenting cells (FIGS. 20 and 21). Asshown in FIG. 18, the binding signal in ELISA correlated to thesecretion level of these clones. As shown in FIGS. 20 and 21, a proteinA-purified bispecific IgG bound to both CD30 and ErbB2 antigens. In suchan ELISA (FIG. 21) it was expected that the monospecific IgGs (which arebi-valent) will show a more intense binding signal, each on its cognateantigen, due to avidity effect. A preliminary cell-ELISA (FIG. 22) showsthat the bispecific antibody secreted by clone D3 stainsantigen-positive cells.

Example 10 Construction of Monospecific Antibodies

First, a mono-specific antibody (T427 IgG) was generated that comprisedthe knobs into hole (KIH) mutation. In order to evaluate the bindingactivity of KIH T427 IgG molecules, ELISA was carried out. The ELISAplate was coated with MBP-CD30 and incubated with T427 KIH IgG (fused toPE38). It was demonstrated that the binding ability of T427 KIH moleculewas similar to the binding of unmodified T427 IgG and T427-PE38 IgG-PE38(FIG. 24).

Subsequently, a mono-specific antibody (T427 IgG) was generated thatcomprised both the KIH mutation and the cysteine mutations in the lightchains as described herein above.

In order to produce the mono-specific T427 antibody, 4 chains wereconstructed: IgL-PE38, IgH-knob, IgH-Cys-hole and IgL-Cys. The presenceof 38 kDa PE38 fused to VL-unmodified light chain provided thepossibility to analyze the pairing of the appropriate heavy and lightchains (analogous to KIH heterodimerization analysis) and the formationof the full-sized mono bi-specific molecule (Figure not shown).

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. A bispecific antibody comprising an Fc region anda Fab region, wherein: (i) said Fc region comprises two non-identicalheavy chains, wherein at least one of said two non-identical heavychains comprises an amino acid modification so as to form a stericcomplementation between said two non-identical heavy chains, whereinsaid Fc region comprises a protuberance of one heavy chain of said Fcregion and a sterically compensatory cavity on a second heavy chain ofsaid Fc region, said protuberance protruding into said compensatorycavity, wherein said complementation is generated by: (a) substitutingan amino acid at one position on a CH3 domain of said one heavy chainwith another amino acid having a larger side chain volume than theoriginal amino acid, said amino acid having a larger side chain beingselected from the group consisting of tyrosine, arginine, phenylalanine,isoleucine and tryptophan so as to generate said protuberance; and (b)substituting an amino acid at one position on a CH3 domain of saidsecond heavy chain with another amino acid having a smaller side chainvolume than the original amino acid so as to generate said compensatorycavity, said amino acid having a smaller side chain being selected fromthe group consisting of alanine, glycine, valine and threonine; and (ii)said Fab region comprises a first covalent link between a first heavychain and a first light chain of said Fab region and a second covalentlink between a second heavy chain and a second light chain of said Fabregion, wherein said first covalent link is a naturally occurringdisulfide bond between a CH1 domain of said one heavy chain and a CLdomain of said one light chain; and said second covalent link is adisulfide bond between a cysteine at position 44 of a V_(H) domain ofsaid second heavy chain and a cysteine at position 100 of a V_(L) domainof said second light chain, wherein the numbering of said position isaccording to Kabat and Wu, wherein said second heavy chain is devoid ofits native disulfide bond with said second light chain, said nativedisulfide bond being an interchain disulfide bond that connects a heavychain to its cognate light chain encoded in a naturally occurringgermline antibody gene.
 2. The antibody of claim 1, being selected fromthe group consisting of a chimeric antibody, a humanized antibody and afully human antibody.
 3. The antibody of claim 1, wherein said CH3domain of said first heavy chain is covalently linked to said CH3 domainof said second heavy chain.
 4. The antibody of claim 1, wherein a firstantigen binding site of the antibody binds a first epitope of an antigenand a second antigen binding site of the antibody binds a second epitopeof said antigen.
 5. The antibody of claim 1, wherein a first antigenbinding site of the antibody binds an epitope of a first antigen and asecond antigen binding site of the antibody binds an epitope of a secondantigen.
 6. The antibody of claim 1, wherein each light chain is linkedto its cognate heavy chain via a single disulfide bond.
 7. The antibodyof claim 1 being an intact antibody.
 8. The antibody of claim 1, whereinthe antibody is selected from the group consisting of IgA, IgD, IgE andIgG.
 9. The antibody of claim 19, wherein said IgG comprises IgG1, IgG2,IgG3 or IgG4.
 10. The antibody of claim 1, wherein said first heavychain comprises a T366W mutation; and said second heavy chain comprisesT366S, L368A, Y407V mutations.
 11. The antibody of claim 21, whereinsaid first heavy chain comprises an S354C mutation and said second heavychain comprises a Y349C mutation.
 12. The antibody of claim 16, whereinsaid first antigen binding site binds CD30 and said second antigenbinding site binds erbB2.
 13. The antibody of claim 16, wherein saidfirst antigen binding site binds CD30 and said second antigen bindingsite binds Pseudomonas Exotoxin (PE).
 14. The antibody of claim 16,wherein said first antigen binding site binds CD30 and said secondantigen binding site binds strepavidin.
 15. The antibody of claim 1,wherein at least one of said heavy chains is attached to a therapeuticmoiety.
 16. The antibody of claim 1, wherein at least one of said heavychains is attached to an identifiable moiety.
 17. The antibody of claim1, being selected from the group consisting of a primate antibody, aporcine antibody, a murine antibody, a bovine antibody, a goat antibodyand an equine antibody.
 18. A method of preparing the antibody of claim1, comprising: (a) providing a first nucleic acid molecule encoding saidfirst heavy chain; (b) providing a second nucleic acid molecule encodingsaid second heavy chain; (c) providing a third nucleic acid moleculeencoding said first light chain; (d) providing a fourth nucleic acidmolecule encoding said second light chain; (e) culturing host cellscomprising said first, second, third and fourth nucleic acid moleculesunder conditions that permit expression of the nucleic acid molecules;and (f) recovering the antibody of claim
 1. 19. The method of claim 29,wherein said host cells comprise bacterial cells.
 20. The method ofclaim 29, wherein said host cells comprise mammalian cells.
 21. Themethod of claim 30, wherein said expression takes place in inclusionbodies of said bacterial cells.
 22. The method of claim 29, wherein eachof said nucleic acid molecules are transfected into different hostcells.
 23. The method of claim 29, wherein each of said nucleic acidmolecules are transfected into the same host cell.
 24. The method ofclaim 30, wherein said bacterial cells comprise gram negative bacterialcells.
 25. The method of claim 29, further comprising purifying theantibody on a protein selected from the group consisting of Protein A,Protein G and Protein L following step (f).
 26. A pharmaceuticalcomposition comprising as an active agent the antibody of claim 1 and apharmaceutically acceptable carrier.
 27. A method of treating aninfection or inflammatory disease or inflammatory disorder in a subjectin need thereof, comprising administering a therapeutically effectiveamount of the antibody of claim 1 to the subject, thereby treating theinfection or inflammatory disease or inflammatory disorder.
 28. Themethod of claim 37, wherein said inflammatory disorder is cancer.